Genetic polymorphisms associated with statin response and cardiovascular diseases, methods of detection and uses thereof

ABSTRACT

The present invention provides compositions and methods based on genetic polymorphisms that are associated with response to statin treatment, particularly for reducing the risk of cardiovascular disease, especially coronary heart disease (such as myocardial infarction) and stroke. For example, the present invention relates to nucleic acid molecules containing the polymorphisms, variant proteins encoded by these nucleic acid molecules, reagents and kits for detecting the polymorphic nucleic acid molecules and variant proteins, and methods of using the nucleic acid molecules and proteins as well as methods of using reagents and kits for their detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. non-provisionalapplication Ser. No. 15/952,792, filed Apr. 13, 2018, which is acontinuation application of U.S. non-provisional application Ser. No.14/834,946, filed Aug. 25, 2015, which is a continuation application ofU.S. non-provisional application Ser. No. 13/833,905, filed Mar. 15,2013, which is a continuation application of U.S. non-provisionalapplication Ser. No. 13/085,955, filed Apr. 13, 2011, which claims thebenefit of U.S. provisional application Ser. No. 61/325,689 filed Apr.19, 2010, U.S. provisional application Ser. No. 61/332,509 filed May 7,2010, and U.S. provisional application Ser. No. 61/405,972 filed Oct.22, 2010, the contents of each of which are hereby incorporated byreference in their entirety into this application.

FIELD OF THE INVENTION

The present invention is in the field of drug response and disease risk,particularly genetic polymorphisms that are associated with response tostatins, especially for the prevention or treatment of cardiovasculardiseases (CVD) such as coronary heart disease (CHD) (which includescoronary events such as myocardial infarction (MI)) and cerebrovascularevents (such as stroke). In particular, the present invention relates tospecific single nucleotide polymorphisms (SNPs) in the human genome, andtheir association with variability in responsiveness to statin treatment(including preventive treatment) in reducing CVD risk between differentindividuals. These SNPs are also useful for assessing an individual'srisk for developing CVD. The SNPs disclosed herein can be used, forexample, as targets for diagnostic reagents and for the development oftherapeutic agents. In particular, the SNPs of the present invention areuseful for such uses as predicting an individual's response totherapeutic agents such as evaluating the likelihood of an individualdifferentially responding positively to statins, particularly for thetreatment or prevention of CVD (particularly CHD such as MI, as well asstroke), identifying an individual who has an increased or decreasedrisk of developing CVD (particularly CHD such as MI, as well as stroke),for early detection of the disease, for providing clinically importantinformation for the prevention and/or treatment of CVD, for predictingrecurrence of CVD, and for screening and selecting therapeutic agents.Methods, assays, kits, and reagents for detecting the presence of thesepolymorphisms and their encoded products are provided.

BACKGROUND OF THE INVENTION

The present invention relates to SNPs that are associated withvariability between individuals in their response to statins,particularly for the prevention or treatment of cardiovascular disease(CVD), which includes coronary heart disease (CHD) (which furtherincludes myocardial infarction (MI) and other coronary events) andcerebrovascular events such as stroke and transient ischemic attack(TIA). These SNPs are also useful for determining an individual's riskfor developing CVD, particularly CHD (including coronary events such asMI) as well as cerebrovascular events (such as stroke and TIA).

HMG-CoA Reductase Inhibitors (Statins)

HMG-CoA reductase inhibitors (statins) are used for the treatment andprevention of CVD, particularly CHD (including coronary events such asMI) and cerebrovascular events (such as stroke). Reduction of MI,stroke, and other coronary and cerebrovascular events and totalmortality by treatment with HMG-CoA reductase inhibitors has beendemonstrated in a number of randomized, double-blinded,placebo-controlled prospective trials (D. D. Waters, Clin Cardiol 24(8Suppl):III3-7 (2001); B. K. Singh and J. L. Mehta, Curr Opin Cardiol17(5):503-11 (2002)). These drugs are thought to typically have theirprimary effect through the inhibition of hepatic cholesterol synthesis,thereby upregulating LDL receptors in the liver. The resultant increasein LDL catabolism results in decreased circulating LDL, a major riskfactor for cardiovascular disease.

Examples of statins include, but are not limited to, atorvastatin(Lipitor®), rosuvastatin (Crestor®), pravastatin (Pravachol®),simvastatin (Zocor®), fluvastatin (Lescol®), and lovastatin (Mevacor®),as well as combination therapies that include a statin such assimvastatin+ezetimibe (Vytorin®), lovastatin+niacin (Advicor®),atorvastatin+amlodipine besylate (Caduet®), and simvastatin+niacin(Simcor®).

Statins can be divided into two types according to their physicochemicaland pharmacokinetic properties. Statins such as atorvastatin,simvastatin, lovastatin, and cerivastatin are lipophilic in nature and,as such, diffuse across membranes and thus are highly cell permeable.Hydrophilic statins such as pravastatin are more polar, such that theyrequire specific cell surface transporters for cellular uptake. K.Ziegler and W. Stunkel, Biochim Biophys Acta 1139(3):203-9 (1992); M.Yamazaki et al., Am J Physiol 264(1 Pt 1):G36-44 (1993); T. Komai etal., Biochem Pharmacol 43(4):667-70 (1992). The latter statins utilizesa transporter, OATP2, whose tissue distribution is confined to the liverand, therefore, they are relatively hepato-specific inhibitors. B.Hsiang et al., J Biol Chem 274(52):37161-37168 (1999). The formerstatins, not requiring specific transport mechanisms, are available toall cells and they can directly impact a much broader spectrum of cellsand tissues. These differences in properties may influence the spectrumof activities that each statin possesses. Pravastatin, for instance, hasa low myopathic potential in animal models and myocyte cultures comparedto lipophilic statins. B. A. Masters et al., Toxicol Appl Pharmacol131(1):163-174 (1995); K. Nakahara et al., Toxicol Appl Pharmacol152(1):99-106 (1998); J. C. Reijneveld et al., Pediatr Res39(6):1028-1035 (1996). Statins are reviewed in Vaughan et al., “Updateon Statins: 2003”, Circulation 2004; 110; 886-892.

Evidence from gene association studies is accumulating to indicate thatresponses to drugs are, indeed, at least partly under genetic control.As such, pharmacogenetics—the study of variability in drug responsesattributed to hereditary factors in different populations—maysignificantly assist in providing answers toward meeting this challenge.A. D. Roses, Nature 405(6788):857-865 (2000); V. Mooser et al., J ThrombHaemost 1(7):1398-1402 (2003); L. M. Humma and S. G. Terra, Am J HealthSyst Pharm 59(13):1241-1252 (2002). Associations have been reportedbetween specific genotypes, as defined by SNPs and other geneticsequence variations, and specific responses to cardiovascular drugs. Forexample, a polymorphism in the KIF6 gene is associated with response tostatin treatment (Iakoubova et al., “Polymorphism in KIF6 gene andbenefit from statins after acute coronary syndromes: results from thePROVE IT-TIMI 22 study”, J Am Coll Cardiol. 2008 Jan. 29; 51(4):449-55;Iakoubova et al., “Association of the 719Arg variant of KIF6 with bothincreased risk of coronary events and with greater response to statintherapy”, J Am Coll Cardiol. 2008 Jun. 3; 51(22):2195; Iakoubova et al.,“KIF6 Trp719Arg polymorphism and the effect of statin therapy in elderlypatients: results from the PROSPER study”, Eur J Cardiovasc PrevRehabil. 2010 Apr. 20; and Shiffman et al., “Effect of pravastatintherapy on coronary events in carriers of the KIF6 719Arg allele fromthe cholesterol and recurrent events trial”, Am J Cardiol. 2010 May 1;105(9):1300-5).

There is a need for genetic markers that can be used to predict anindividual's responsiveness to statins. For example, there is a growingneed to better identify people who have a high chance of benefiting fromstatins, and those who have a low risk of developing side-effects. Forexample, severe myopathies represent a significant risk for a lowpercentage of the patient population, and this may be a particularconcern for patients who are treated more aggressively with statins.Furthermore, different patients may have the same the risk for adverseevents but are more likely to benefit from a drug (such as statins) andthis may justify use of the drug in those individuals who are morelikely to benefit. Similarly, in individuals who are less likely tobenefit from a drug but are at risk for adverse events, use of the drugin these individuals can be de-prioritized or delayed.

An example of a large trial which analyzed the benefits of statintreatment for reducing the risk of CVD in a large population was theJUPITER Study (described in Ridker et al., “Rosuvastatin to preventvascular events in men and women with elevated C-reactive protein”, NEngl J Med. 2008 Nov. 20; 359(21):2195-207), which demonstrated thatrosuvastatin (Crestor®) significantly reduced the incidence of majorcardiovascular events (including MI, stroke, arterial revascularization,hospitalization for unstable angina, and death from cardiovascularcauses) in a study of 17,802 individuals.

The benefits of using statins for stroke is also described in O'Regan etal., “Statin therapy in stroke prevention: a meta-analysis involving121,000 patients”, Am J Med. 2008 January; 121(1):24-33 and Everett etal., “Rosuvastatin in the prevention of stroke among men and women withelevated levels of C-reactive protein: justification for the Use ofStatins in Prevention: an Intervention Trial Evaluating Rosuvastatin(JUPITER)”, Circulation. 2010 Jan. 5; 121(1):143-50.

Cardiovascular Disease (CVD), Including Coronary Heart Disease (CHD) andStroke

Cardiovascular disease (CVD) includes coronary heart disease (CHD)(which further includes myocardial infarction (MI) and other coronaryevents) and cerebrovascular events such as stroke and transient ischemicattack (TIA).

Coronary heart disease (CHD) is defined herein as encompassing MI (fatalor non-fatal) and other coronary events, death from coronary disease,angina pectoris (particularly unstable angina), and coronary stenosis.The presence of CHD may be indicated by the occurrence of medicalinterventions such as coronary revascularization, which can includepercutaneous transluminal coronary angioplasty (PTCA), coronary stentplacement, and coronary artery bypass graft (CABG). Cardiovasculardisease (CVD) is defined herein as encompassing CHD as well ascerebrovascular events such as stroke and transient ischemic attack(TIA).

Myocardial Infarction (MI)

Myocardial infarction (MI) is encompassed within CHD. MI, also referredto as a “heart attack”, is the most common cause of mortality indeveloped countries. The incidence of MI is still high despite currentlyavailable preventive measures and therapeutic intervention. More than1,500,000 people in the U.S. suffer acute MI each year, many withoutseeking help due to unrecognized MI, and one third of these people die.The lifetime risk of coronary artery disease events at age 40 is 42.4%for men, nearly one in two, and 24.9% for women, or one in four (D. M.Lloyd-Jones, Lancet 353:89-92 (1999)).

MI is a multifactorial disease that involves atherogenesis, thrombusformation and propagation. Thrombosis can result in complete or partialocclusion of coronary arteries. The luminal narrowing or blockage ofcoronary arteries reduces oxygen and nutrient supply to the cardiacmuscle (cardiac ischemia), leading to myocardial necrosis and/orstunning. MI, unstable angina, and sudden ischemic death are clinicalmanifestations of cardiac muscle damage. All three endpoints are part ofacute coronary syndrome since the underlying mechanisms of acutecomplications of atherosclerosis are considered to be the same.

Atherogenesis, the first step of pathogenesis of MI, is an interactionbetween blood elements, mechanical forces, disturbed blood flow, andvessel wall abnormality that results in plaque accumulation. An unstable(vulnerable) plaque is an underlying cause of arterial thrombotic eventsand MI. A vulnerable plaque is a plaque, often not stenotic, that has ahigh likelihood of becoming disrupted or eroded, thus forming athrombogenic focus. The “vulnerability” of an individual to MI may bedue to vulnerable plaque, blood vulnerability (hypercoagulation,hypothrombolysis), and heart vulnerability (sensitivity of the heart toischemia or propensity for arrhythmia). Recurrent myocardial infarction(RMI) can generally be viewed as a severe form of MI progression causedby multiple vulnerable plaques that are able to undergo pre-rupture or apre-erosive state, coupled with extreme blood coagulability.

The current diagnosis of MI with presentation (rather than to predict ifMI is likely to occur in the future) is based on the levels of troponinI or T that indicate the cardiac muscle progressive necrosis, impairedelectrocardiogram (ECG), and detection of abnormal ventricular wallmotion or angiographic data (the presence of acute thrombi). However,due to the asymptomatic nature of 25% of acute MIs (absence of atypicalchest pain, low ECG sensitivity), a significant portion of MIs are notdiagnosed and therefore not treated appropriately (e.g., prevention ofrecurrent MIs).

MI risk assessment and prognosis is currently done using classic riskfactors or the recently introduced Framingham Risk Index. Both of theseassessments put a significant weight on LDL levels to justify preventivetreatment. However, it is well established that half of all MIs occur inindividuals without overt hyperlipidemia.

Other emerging risk factors of MI are inflammatory biomarkers such asC-reactive protein (CRP), ICAM-1, SAA, TNF α, homocysteine, impairedfasting glucose, new lipid markers (ox LDL, Lp-a, MAD-LDL, etc.) andpro-thrombotic factors (fibrinogen, PAI-1). These markers havesignificant limitations such as low specificity and low positivepredictive value, and the need for multiple reference intervals to beused for different groups of people (e.g., males-females, smokers-nonsmokers, hormone replacement therapy users, different age groups). Theselimitations diminish the utility of such markers as independentprognostic markers for MI screening.

Genetics plays an important role in MI risk. Families with a positivefamily history of MI account for 14% of the general population, 72% ofpremature MIs, and 48% of all MIs (R. R. Williams, Am J Cardiology87:129 (2001)). Associations have been reported between geneticpolymorphisms and MI risk. For example, polymorphism in the KIF6, LPA,and other genes and chromosomal regions are associated with MI risk(Shiffman et al., “Association of gene variants with incident myocardialinfarction in the Cardiovascular Health Study”, Arterioscler Thromb VascBiol. 2008 January; 28(1):173-9; Bare et al., “Five common gene variantsidentify elevated genetic risk for coronary heart disease”, Genet Med.2007 October; 9(10):682-9; Iakoubova et al., “Association of theTrp719Arg polymorphism in kinesin-like protein 6 with myocardialinfarction and coronary heart disease in 2 prospective trials: the CAREand WOSCOPS trials”, J Am Coll Cardiol. 2008 Jan. 29; 51(4):435-43; andShiffman et al., “A kinesin family member 6 variant is associated withcoronary heart disease in the Women's Health Study”, J Am Coll Cardiol.2008 Jan. 29; 51(4):444-8.

Genetic markers such as single nucleotide polymorphisms (SNPs) arepreferable to other types of biomarkers. Genetic markers that areprognostic for MI can be genotyped early in life and could predictindividual response to various risk factors. The combination of serumprotein levels and genetic predisposition revealed by genetic analysisof susceptibility genes can provide an integrated assessment of theinteraction between genotypes and environmental factors, resulting insynergistically increased prognostic value of diagnostic tests.

Thus, there is an urgent need for novel genetic markers that arepredictive of predisposition to CHD such as MI, particularly forindividuals who are unrecognized as having a predisposition to MI. Suchgenetic markers may enable prognosis of MI in much larger populationscompared with the populations that can currently be evaluated by usingexisting risk factors and biomarkers. The availability of a genetic testmay allow, for example, appropriate preventive treatments for acutecoronary events to be provided for susceptible individuals (suchpreventive treatments may include, for example, statin treatments andstatin dose escalation, as well as changes to modifiable risk factors),lowering of the thresholds for ECG and angiography testing, and allowadequate monitoring of informative biomarkers. Moreover, the discoveryof genetic markers associated with MI can provide novel targets fortherapeutic intervention or preventive treatments of MI, and enable thedevelopment of new therapeutic agents for treating or preventing MI andother cardiovascular disorders.

Furthermore, novel genetic markers that are predictive of predispositionto MI can be particularly useful for identifying individuals who are atrisk for early-onset MI. “Early-onset MI” may be defined as MI in menwho are less than 55 years of age and women who are less than 65 yearsof age (K. O. Akosah et al., “Preventing myocardial infarction in theyoung adult in the first place: How do the National CholesterolEducation Panel III guidelines perform?” JACC 41(9):1475-1479 (2003)).Individuals who experience early-onset MI may not be effectivelyidentified by current cholesterol treatment guidelines, such as thosesuggested by the National Cholesterol Education Program. In one study,for example, a significant number of individuals who suffered MI at anearlier age 50 years) were shown to have LDL cholesterol below 100 mg/dl(K. O. Akosah et al., “Myocardial infarction in young adults withlow-density lipoprotein cholesterol levels less than or equal to 100mg/dl. Clinical profile and 1-year outcomes.” Chest 120:1953-1958(2001)). Because risk for MI can be reduced by lifestyle changes and bytreatment of modifiable risk factors, better methods to identifyindividuals at risk for early-onset MI could be useful for makingpreventive treatment decisions, especially considering that thesepatients may not be identified for medical management by conventionaltreatment guidelines. Genetic markers for risk of early-onset MI couldpotentially be incorporated into individual risk assessment protocols,as they have the advantage of being easily detected at any age.

Stroke

Stroke is a prevalent and serious cerebrovascular disease. It affects4.7 million individuals in the United States, with 500,000 first attacksand 200,000 recurrent cases yearly. Approximately one in four men andone in five women aged 45 years will have a stroke if they live to their85th year. About 25% of those who have a stroke die within a year.Stroke is the third leading cause of mortality in the United States andis responsible for 170,000 deaths a year. Among those who survive astroke attack, 30 to 50% do not regain functional independence. Stroketherefore is the most common cause of disability and the second leadingcause of dementia (Heart Disease and Stroke Statistics—2004 Update,American Heart Association).

Stroke occurs when an artery bringing oxygen and nutrients to the braineither ruptures, causing hemorrhagic stroke, or gets occluded, causingischemic stroke. Ischemic stroke can be caused by thrombi formation atthe site of an atherosclerotic plaque rupture (this type of ischemicstroke is interchangeably referred to as thrombotic or atherothromboticstroke) or by emboli (clots) that have travelled from another part ofthe vasculature (this type of ischemic stroke is referred to as embolicstroke), often from the heart (this type of embolic stroke may bereferred to as cardioembolic stroke). In both ischemic and hemorrhagicstroke, a cascade of cellular changes due to ischemia or increasedcranial pressure leads to injuries or death of the brain cells. In theUnited States, the majority (about 80-90%) of stroke cases are ischemic(Rathore, et al., Stroke 33:2718-2721 ((2002)), including 30%large-vessel thrombotic (also referred to as large-vessel occlusivedisease), 20% small-vessel thrombotic (also referred to as small-vesselocclusive disease), and 30% embolic or cardiogenic (caused by a clotoriginating from elsewhere in the body, e.g., from blood pooling due toatrial fibrillation, or from carotid artery stenosis). The ischemic formof stroke results from obstruction of blood flow in cerebral bloodvessels, and it shares common pathological etiology with atherosclerosisand thrombosis.

About 10-20% of stroke cases are of the hemorrhagic type (Rathore, etal., Stroke 33:2718-2721 ((2002)), involving bleeding within or aroundthe brain. Bleeding within the brain is known as cerebral hemorrhage,which is often linked to high blood pressure. Bleeding into the meningessurrounding the brain is known as a subarachnoid hemorrhage, which couldbe caused by a ruptured cerebral aneurysm, an arteriovenousmalformation, or a head injury. The hemorrhagic stroke, although lessprevalent, poses a greater danger. Whereas about 8% of ischemic strokecases result in death within 30 days, about 38% of hemorrhagic strokecases result in death within the same time period (Collins, et al., J.Clin. Epidemiol. 56:81-87 (2003)).

Transient ischemic attack (TIA) is a condition related to stroke.According to the National Institute of Neurological Disorders and Stroke(NINDS), “A transient ischemic attack (TIA) is a transient stroke thatlasts only a few minutes. It occurs when the blood supply to part of thebrain is briefly interrupted. TIA symptoms, which usually occursuddenly, are similar to those of stroke but do not last as long. Mostsymptoms of a TIA disappear within an hour, although they may persistfor up to 24 hours. Symptoms can include: numbness or weakness in theface, arm, or leg, especially on one side of the body; confusion ordifficulty in talking or understanding speech; trouble seeing in one orboth eyes; and difficulty with walking, dizziness, or loss of balanceand coordination”. NINDS further states that, “TIAs are often warningsigns that a person is at risk for a more serious and debilitatingstroke. About one-third of those who have a TIA will have an acutestroke some time in the future. Many strokes can be prevented by heedingthe warning signs of TIAs and treating underlying risk factors.”

Known risk factors for stroke or TIA can be divided into modifiable andnon-modifiable risk factors. Older age, male sex, black or Hispanicethnicity, and family history of stroke are non-modifiable risk factors.Modifiable risk factors include hypertension, smoking, increased insulinlevels, asymptomatic carotid disease, cardiac vessel disease, andhyperlipidemia.

Multiple reports based on twin studies (Brass et al., Stroke. 1992;23:221-223 and Bak et al., Stroke. 2002; 33:769-774) and family studies(Welin L, et al. N Engl J Med. 1987; 317:521-526 and Jousilahti et al.,Stroke. 1997; 28:1361-136) have shown that genetics contributes to riskof stroke independently of traditional risk factors. A number of geneticmarkers have been reported to be associated with stroke. For example,SNPs in the 4q25 region were reported to be associated with stroke(Gretarsdottir et al., “Risk variants for atrial fibrillation onchromosome 4q25 associate with ischemic stroke”, Ann Neurol. 2008;64:402-409) and with atrial fibrillation (AF) (Gudbjartsson et al.,“Variants conferring risk of atrial fibrillation on chromosome 4q25”,Nature. 2007; 448:353-357), SNPs in the 16q22 region (Gudbjartsson etal., “A sequence variant in ZFHX3 on 16q22 associates with atrialfibrillation and ischemic stroke”, Nat Genet. 2009; 41:876-878) and inthe 9p21 region were found to be associated with noncardioembolic oratherothrombotic stroke (Luke et al., “Polymorphisms associated withboth noncardioembolic stroke and coronary heart disease: vienna strokeregistry”, Cerebrovasc Dis. 2009; 28:499-504 and Gschwendtner et al.,“Sequence variants on chromosome 9p21.3 confer risk for atheroscleroticstroke”, Ann Neurol. 2009; 65:531-539), and variants in the 12p13 regionwere associated with stroke in general and with atherothrombotic strokein particular (Ikram et al., “Genomewide association studies of stroke”,N Engl J Med. 2009; 360:1718-1728).

The acute nature of stroke leaves physicians with little time to preventor lessen the devastation of brain damage. Strategies to diminish theimpact of stroke include prevention and treatment with thrombolytic and,possibly, neuroprotective agents. The success of preventive measureswill depend on the identification of risk factors in individual patientsand means to modulate their impact.

Although some risk factors for stroke or TIA are not modifiable, such asage and family history, other underlying pathology or risk factors ofstroke or TIA such as atherosclerosis, hypertension, smoking, diabetes,aneurysm, and atrial fibrillation, are chronic and amenable to effectivelife-style changes, pharmacological interventions, as well as surgicaltreatments. Early recognition of patients with informative risk factors,and especially those with a family history, using a non-invasive test ofgenetic markers associated with stroke can enable physicians to targetthe highest risk individuals for aggressive risk reduction.

Thus, there is a need for the identification of genetic markers that arepredictive of an individual's predisposition to stroke or TIA and othervascular diseases. Furthermore, the identification of genetic markerswhich are useful for identifying individuals who are at an increasedrisk of having a stroke may lead to, for example, better preventive andtherapeutic strategies, economic models, and health care policydecisions.

Single Nucleotide Polymorphisms (SNPs)

The genomes of all organisms undergo spontaneous mutations in the courseof their continuing evolution, generating variant forms of progenitorgenetic sequences. Gusella, Ann Rev Biochem 55:831-854 (1986). A variantform may confer an evolutionary advantage or disadvantage relative to aprogenitor form or may be neutral. In some instances, a variant formconfers an evolutionary advantage to individual members of a species andis eventually incorporated into the DNA of many or most members of thespecies and effectively becomes the progenitor form. Additionally, theeffects of a variant form may be both beneficial and detrimental,depending on the environment. For example, a heterozygous sickle cellmutation confers resistance to malaria, but a homozygous sickle cellmutation is usually lethal. In many cases, both progenitor and variantforms survive and co-exist in a species population. The coexistence ofmultiple forms of a genetic sequence segregating at appreciablefrequencies is defined as a genetic polymorphism, which includes singlenucleotide polymorphisms (SNPs).

Approximately 90% of all genetic polymorphisms in the human genome areSNPs. SNPs are single base positions in DNA at which different alleles,or alternative nucleotides, exist in a population. The SNP position(interchangeably referred to herein as SNP, SNP site, SNP locus, SNPmarker, or marker) is usually preceded by and followed by highlyconserved sequences (e.g., sequences that vary in less than 1/100 or1/1000 members of the populations). An individual may be homozygous orheterozygous for an allele at each SNP position. A SNP can, in someinstances, be referred to as a “cSNP” to denote that the nucleotidesequence containing the SNP is an amino acid coding sequence.

A SNP may arise from a substitution of one nucleotide for another at thepolymorphic site. Substitutions can be transitions or transversions. Atransition is the replacement of one purine nucleotide by another purinenucleotide, or one pyrimidine by another pyrimidine. A transversion isthe replacement of a purine by a pyrimidine, or vice versa. A SNP mayalso be a single base insertion or deletion variant referred to as an“indel.” Weber et al., “Human diallelic insertion/deletionpolymorphisms,” Am J Hum Genet 71(4):854-62 (October 2002).

A synonymous codon change, or silent mutation/SNP (terms such as “SNP”,“polymorphism”, “mutation”, “mutant”, “variation”, and “variant” areused herein interchangeably), is one that does not result in a change ofamino acid due to the degeneracy of the genetic code. A substitutionthat changes a codon coding for one amino acid to a codon coding for adifferent amino acid (i.e., a non-synonymous codon change) is referredto as a missense mutation. A nonsense mutation results in a type ofnon-synonymous codon change in which a stop codon is formed, therebyleading to premature termination of a polypeptide chain and a truncatedprotein. A read-through mutation is another type of non-synonymous codonchange that causes the destruction of a stop codon, thereby resulting inan extended polypeptide product. While SNPs can be bi-, tri-, ortetra-allelic, the vast majority of SNPs are bi-allelic, and are thusoften referred to as “bi-allelic markers,” or “di-allelic markers.”

As used herein, references to SNPs and SNP genotypes include individualSNPs and/or haplotypes, which are groups of SNPs that are generallyinherited together. Haplotypes can have stronger correlations withdiseases or other phenotypic effects compared with individual SNPs, andtherefore may provide increased diagnostic accuracy in some cases.Stephens et al., Science 293:489-493 (July 2001).

Causative SNPs are those SNPs that produce alterations in geneexpression or in the expression, structure, and/or function of a geneproduct, and therefore are most predictive of a possible clinicalphenotype. One such class includes SNPs falling within regions of genesencoding a polypeptide product, i.e. cSNPs. These SNPs may result in analteration of the amino acid sequence of the polypeptide product (i.e.,non-synonymous codon changes) and give rise to the expression of adefective or other variant protein. Furthermore, in the case of nonsensemutations, a SNP may lead to premature termination of a polypeptideproduct. Such variant products can result in a pathological condition,e.g., genetic disease. Examples of genes in which a SNP within a codingsequence causes a genetic disease include sickle cell anemia and cysticfibrosis.

Causative SNPs do not necessarily have to occur in coding regions;causative SNPs can occur in, for example, any genetic region that canultimately affect the expression, structure, and/or activity of theprotein encoded by a nucleic acid. Such genetic regions include, forexample, those involved in transcription, such as SNPs in transcriptionfactor binding domains, SNPs in promoter regions, in areas involved intranscript processing, such as SNPs at intron-exon boundaries that maycause defective splicing, or SNPs in mRNA processing signal sequencessuch as polyadenylation signal regions. Some SNPs that are not causativeSNPs nevertheless are in close association with, and therefore segregatewith, a disease-causing sequence. In this situation, the presence of aSNP correlates with the presence of, or predisposition to, or anincreased risk in developing the disease. These SNPs, although notcausative, are nonetheless also useful for diagnostics, diseasepredisposition screening, and other uses.

An association study of a SNP and a specific disorder involvesdetermining the presence or frequency of the SNP allele in biologicalsamples from individuals with the disorder of interest, such as CVD, andcomparing the information to that of controls (i.e., individuals who donot have the disorder; controls may be also referred to as “healthy” or“normal” individuals) who are preferably of similar age and race. Theappropriate selection of patients and controls is important to thesuccess of SNP association studies. Therefore, a pool of individualswith well-characterized phenotypes is extremely desirable.

A SNP may be screened in diseased tissue samples or any biologicalsample obtained from a diseased individual, and compared to controlsamples, and selected for its increased (or decreased) occurrence in aspecific pathological condition, such as pathologies related to CVD andin particular, CHD (e.g., MI). Once a statistically significantassociation is established between one or more SNP(s) and a pathologicalcondition (or other phenotype) of interest, then the region around theSNP can optionally be thoroughly screened to identify the causativegenetic locus/sequence(s) (e.g., causative SNP/mutation, gene,regulatory region, etc.) that influences the pathological condition orphenotype. Association studies may be conducted within the generalpopulation and are not limited to studies performed on relatedindividuals in affected families (linkage studies).

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. There is a continuing need toimprove pharmaceutical agent design and therapy. In that regard, SNPscan be used to identify patients most suited to therapy with particularpharmaceutical agents (this is often termed “pharmacogenomics”).Similarly, SNPs can be used to exclude patients from certain treatmentdue to the patient's increased likelihood of developing toxic sideeffects or their likelihood of not responding to the treatment.Pharmacogenomics can also be used in pharmaceutical research to assistthe drug development and selection process. Linder et al., ClinicalChemistry 43:254 (1997); Marshall, Nature Biotechnology 15:1249 (1997);International Patent Application WO 97/40462, Spectra Biomedical; andSchafer et al., Nature Biotechnology 16:3 (1998).

SUMMARY OF THE INVENTION

Exemplary embodiments of the present invention relate to theidentification of SNPs, as well as unique combinations of such SNPs andhaplotypes of SNPs, that are associated with variability betweenindividuals in their response to statins, particularly for theprevention or treatment of cardiovascular disease (CVD), which includescoronary heart disease (CHD) (which further includes myocardialinfarction (MI) and other coronary events) and cerebrovascular eventssuch as stroke. These SNPs are also useful for determining anindividual's risk for developing CVD, particularly CHD (includingcoronary events such as MI) as well as cerebrovascular events such asstroke. The polymorphisms disclosed herein are directly useful astargets for the design of diagnostic and prognostic reagents and thedevelopment of therapeutic and preventive agents for use in thediagnosis, prognosis, treatment, and/or prevention of CVD (particularlyCHD, such as MI), as well as for predicting a patient's response totherapeutic agents such as statins, particularly for the treatment orprevention of CVD (particularly CHD, such as MI).

Based on the identification of SNPs associated with variability betweenindividuals in their response to statins, particularly for reducing therisk of CVD such as CHD (e.g., MI) and stroke, exemplary embodiments ofthe present invention also provide methods of detecting these variantsas well as the design and preparation of detection reagents needed toaccomplish this task. The invention specifically provides, for example,SNPs associated with responsiveness to statin treatment, isolatednucleic acid molecules (including DNA and RNA molecules) containingthese SNPs, variant proteins encoded by nucleic acid moleculescontaining such SNPs, antibodies to the encoded variant proteins,computer-based and data storage systems containing the novel SNPinformation, methods of detecting these SNPs in a test sample, methodsof identifying individuals who have an altered (i.e., increased ordecreased) risk of developing CVD (such as CHD (e.g., MI) or stroke),methods for determining the risk of an individual for recurring CVD(e.g., recurrent MI), methods of treating an individual who has anincreased risk for CVD and/or increased likelihood of responding tostatin treatment, and methods for identifying individuals (e.g.,determining a particular individual's likelihood) who have an altered(i.e., increased or decreased) likelihood of responding to drugtreatment (especially statin treatment), particularly drug treatment ofCVD (e.g., prevention or treatment of CHD such as MI), based on thepresence or absence of one or more particular nucleotides (alleles) atone or more SNP sites disclosed herein or the detection of one or moreencoded variant products (e.g., variant mRNA transcripts or variantproteins), methods of screening for compounds useful in the treatment orprevention of CVD, compounds identified by these methods, methods oftreating or preventing CVD, etc.

Exemplary embodiments of the present invention further provide methodsfor selecting or formulating a treatment regimen (e.g., methods fordetermining whether or not to administer statin treatment to anindividual having CVD, or who is at risk for developing CVD in thefuture, or who has previously had CVD, methods for selecting aparticular statin-based treatment regimen such as dosage and frequencyof administration of statin, or a particular form/type of statin such asa particular pharmaceutical formulation or statin compound, methods foradministering (either in addition to or instead of a statin) analternative, non-statin-based treatment, such as niacin, fibrates, orezetimibe (e.g., Zetia® or Ezetrol®), to individuals who are predictedto be unlikely to respond positively to statin treatment, etc.), andmethods for determining the likelihood of experiencing toxicity or otherundesirable side effects from statin treatment, etc. Various embodimentsof the present invention also provide methods for selecting individualsto whom a statin or other therapeutic will be administered based on theindividual's genotype, and methods for selecting individuals for aclinical trial of a statin or other therapeutic agent based on thegenotypes of the individuals (e.g., selecting individuals to participatein the trial who are most likely to respond positively from the statintreatment and/or excluding individuals from the trial who are unlikelyto respond positively from the statin treatment based on their SNPgenotype(s), or selecting individuals who are unlikely to respondpositively to statins based on their SNP genotype(s) to participate in aclinical trial of another type of drug that may benefit them). Furtherembodiments of the present invention provide methods for reducing anindividual's risk of developing CVD (such as CHD (e.g., MI) or stroke)using statin treatment, including preventing recurring CVD (e.g.,recurrent MI) using statin treatment, when said individual carries oneor more SNPs identified herein as being associated with statin response.

Tables 1 and 2 provides gene information, references to theidentification of transcript sequences (SEQ ID NOS:1-51), encoded aminoacid sequences (SEQ ID NOS:52-102), genomic sequences (SEQ IDNOS:177-622), transcript-based context sequences (SEQ ID NOS:103-176)and genomic-based context sequences (SEQ ID NOS:623-3661) that containthe SNPs of the present application, and extensive SNP information thatincludes observed alleles, allele frequencies, populations/ethnic groupsin which alleles have been observed, information about the type of SNPand corresponding functional effect, and, for cSNPs, information aboutthe encoded polypeptide product. The actual transcript sequences (SEQ IDNOS:1-51), amino acid sequences (SEQ ID NOS:52-102), genomic sequences(SEQ ID NOS:177-622), transcript-based SNP context sequences (SEQ IDNOS:103-176), and genomic-based SNP context sequences (SEQ IDNOS:623-3661) are provided in the Sequence Listing.

In certain exemplary embodiments, the invention provides methods foridentifying an individual who has an altered likelihood of responding tostatin treatment or an altered risk for developing CVD, particularly CHDor stroke (including, for example, a first incidence and/or a recurrenceof the disease, such as primary or recurrent MI), in which the methodcomprises detecting a single nucleotide polymorphism (SNP) in any one ofthe nucleotide sequences of SEQ ID NOS:1-51, SEQ ID NOS:103-176, SEQ IDNOS:177-622, and SEQ ID NOS:623-3661 in said individual's nucleic acids,wherein the SNP is specified in Table 1 and/or Table 2, and the presenceof the SNP is indicative of an altered response to statin treatment ofan altered risk for CVD in said individual. In certain embodiments, theCVD is CHD, particularly MI. In certain other embodiments, the CVD isstroke. In certain exemplary embodiments of the invention, SNPs thatoccur naturally in the human genome are provided within isolated nucleicacid molecules. These SNPs are associated with response to statintreatment thereby reducing the risk of CVD, such as CHD (e.g., MI) orstroke, such that they can have a variety of uses in the diagnosis,prognosis, treatment, and/or prevention of CVD, and particularly in thetreatment or prevention of CVD using statins. In certain embodiments, anucleic acid of the invention is an amplified polynucleotide, which isproduced by amplification of a SNP-containing nucleic acid template. Inanother embodiment, the invention provides for a variant protein that isencoded by a nucleic acid molecule containing a SNP disclosed herein.

In further embodiments of the invention, reagents for detecting a SNP inthe context of its naturally-occurring flanking nucleotide sequences(which can be, e.g., either DNA or mRNA) are provided. In particular,such a reagent may be in the form of, for example, a hybridization probeor an amplification primer that is useful in the specific detection of aSNP of interest. In an alternative embodiment, a protein detectionreagent is used to detect a variant protein that is encoded by a nucleicacid molecule containing a SNP disclosed herein. A preferred embodimentof a protein detection reagent is an antibody or an antigen-reactiveantibody fragment. Various embodiments of the invention also providekits comprising SNP detection reagents, and methods for detecting theSNPs disclosed herein by employing the SNP detection reagents. Anexemplary embodiment of the present invention provides a kit comprisinga SNP detection reagent for use in determining whether a human's riskfor CVD is reduced by treatment with statins based upon the presence orabsence of a particular allele of one or more SNPs disclosed herein.

In various embodiments, the present invention provides methods forevaluating whether an individual is likely (or unlikely) to respond tostatin treatment (i.e., benefit from statin treatment)), particularlystatin treatment for reducing the risk of CVD, particularly CHD (such asMI) or stroke, by detecting the presence or absence of one or more SNPalleles disclosed herein. The present invention also provides methods ofidentifying an individual having an increased or decreased risk ofdeveloping CVD, such as CHD (e.g., MI) or stroke, by detecting thepresence or absence of one or more SNP alleles disclosed herein.

In certain embodiments, the presence of a statin response alleledisclosed herein in Tables 4-22 (an allele associated with increasedresponse to statin treatment for reducing CVD or CHD risk) is detectedand indicates that an individual has an increased risk for developingCVD, such as CHD (e.g., MI) or stroke. In these embodiments, in whichthe same allele is associated with both increased risk for developingCVD and increased response to statin treatment (i.e., the same allele isboth a risk and a response allele), this increased risk for developingCVD can be reduced by administering statin treatment to an individualhaving the allele.

The nucleic acid molecules of the invention can be inserted in anexpression vector, such as to produce a variant protein in a host cell.Thus, the present invention also provides for a vector comprising aSNP-containing nucleic acid molecule, genetically-engineered host cellscontaining the vector, and methods for expressing a recombinant variantprotein using such host cells. In another specific embodiment, the hostcells, SNP-containing nucleic acid molecules, and/or variant proteinscan be used as targets in a method for screening and identifyingtherapeutic agents or pharmaceutical compounds useful in the treatmentor prevention of CVD, such as CHD (e.g., MI) or stroke.

An aspect of this invention is a method for treating or preventing CVDsuch as CHD or stroke (including, for example, a first occurrence and/ora recurrence of the disease, such as primary or recurrent MI), in ahuman subject wherein said human subject harbors a SNP, gene,transcript, and/or encoded protein identified in Tables 1 and 2, whichmethod comprises administering to said human subject a therapeuticallyor prophylactically effective amount of one or more agents counteractingthe effects of the disease, such as by inhibiting (or stimulating) theactivity of a gene, transcript, and/or encoded protein identified inTables 1 and 2.

Another aspect of this invention is a method for identifying an agentuseful in therapeutically or prophylactically treating CVD (particularlyCHD or stroke), in a human subject wherein said human subject harbors aSNP, gene, transcript, and/or encoded protein identified in Tables 1 and2, which method comprises contacting the gene, transcript, or encodedprotein with a candidate agent under conditions suitable to allowformation of a binding complex between the gene, transcript, or encodedprotein and the candidate agent and detecting the formation of thebinding complex, wherein the presence of the complex identifies saidagent.

Another aspect of this invention is a method for treating or preventingCVD such as CHD (e.g., MI) or stroke, in a human subject, in which themethod comprises:

-   -   (i) determining that said human subject harbors a SNP, gene,        transcript, and/or encoded protein identified in Tables 1 and 2,        and    -   (ii) administering to said subject a therapeutically or        prophylactically effective amount of one or more agents        counteracting the effects of the disease, such as statins.

Another aspect of the invention is a method for identifying a human whois likely to benefit from statin treatment, in which the methodcomprises detecting an allele of one or more SNPs disclosed herein insaid human's nucleic acids, wherein the presence of the allele indicatesthat said human is likely to benefit from statin treatment.

Another aspect of the invention is a method for identifying a human whois likely to benefit from statin treatment, in which the methodcomprises detecting an allele of one or more SNPs that are in LD withone or more SNPs disclosed herein in said human's nucleic acids, whereinthe presence of the allele of the LD SNP indicates that said human islikely to benefit from statin treatment.

Many other uses and advantages of the present invention will be apparentto those skilled in the art upon review of the detailed description ofthe exemplary embodiments herein. Solely for clarity of discussion, theinvention is described in the sections below by way of non-limitingexamples.

Description of the Text (ASCII) Files Submitted Electronically ViaEFS-Web

The following three text (ASCII) files are submitted electronically viaEFS-Web as part of the instant application:

-   -   1) File SEQLIST_CD000027ORD.txt provides the Sequence Listing.        The Sequence Listing provides the transcript sequences (SEQ ID        NOS:1-51) and protein sequences (SEQ ID NOS:52-102) as referred        to in Table 1, and genomic sequences (SEQ ID NOS:177-622) as        referred to in Table 2, for each gene (or genomic region for        intergenic SNPs) that contains one or more statin        response-associated SNPs of the present invention. Also provided        in the Sequence Listing are context sequences flanking each SNP,        including both transcript-based context sequences as referred to        in Table 1 (SEQ ID NOS:103-176) and genomic-based context        sequences as referred to in Table 2 (SEQ ID NOS:623-3661). The        context sequences generally provide 100 bp upstream (5′) and 100        bp downstream (3′) of each SNP, with the SNP in the middle of        the context sequence, for a total of 200 bp of context sequence        surrounding each SNP. File SEQLIST_CD000027ORD.txt is 63,960 KB        in size, and was created on Apr. 5, 2011.    -   2) File TABLE1_CD27_TRIMMED.TXT provides Table 1, which is 8 KB        in size and was created on Nov. 5, 2015.    -   3) File TABLE2_CD27_TRIMMED.TXT provides Table 2, which is 63 KB        in size and was created on Nov. 5, 2015.

These three text files are hereby incorporated by reference pursuant to37 CFR 1.77(b)(4).

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US11827937B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Description of Table 1 and Table 2

Table 1 and Table 2 (both submitted electronically via EFS-Web as partof the instant application) disclose the SNP and associatedgene/transcript/protein information and sequences for the SNPs disclosedin Tables 4-22, as well as for the LD SNPs disclosed in Table 3. Table 1is based on transcript and protein sequences, whereas Table 2 is basedon genomic sequences.

For each gene, Table 1 provides a header containing gene, transcript andprotein information, followed by a transcript and protein sequenceidentifier (SEQ ID NO), and then SNP information regarding each SNPfound in that gene/transcript including the transcript context sequence.For each gene in Table 2, a header is provided that contains gene andgenomic information, followed by a genomic sequence identifier (SEQ IDNO) and then SNP information regarding each SNP found in that gene,including the genomic context sequence.

Note that SNP markers may be included in both Table 1 and Table 2; Table1 presents the SNPs relative to their transcript sequences and encodedprotein sequences, whereas Table 2 presents the SNPs relative to theirgenomic sequences. In some instances Table 2 may also include, after thelast gene sequence, genomic sequences of one or more intergenic regions,as well as SNP context sequences and other SNP information for any SNPsthat lie within these intergenic regions. Additionally, in either Table1 or 2, a “Related Interrogated SNP” may be listed following a SNP whichis determined to be in LD with that interrogated SNP according to thegiven Power value. SNPs can be readily cross-referenced between allTables based on their Celera hCV (or, in some instances, hDV)identification numbers and/or public rs identification numbers, and tothe Sequence Listing based on their corresponding SEQ ID NOs.

The gene/transcript/protein information includes:

-   -   a gene number (1 through n, where n=the total number of genes in        the Table),    -   a gene symbol, along with an Entrez gene identification number        (Entrez Gene database, National Center for Biotechnology        Information (NCBI), National Library of Medicine (NLM), National        Institutes of Health (NIH))    -   a gene name,    -   an accession number for the transcript (e.g., RefSeq NM number,        or a Celera hCT identification number if no RefSeq NM number is        available) (Table 1 only),    -   an accession number for the protein (e.g., RefSeq NP number, or        a Celera hCP identification number if no RefSeq NP number is        available) (Table 1 only),    -   the chromosome number of the chromosome on which the gene is        located,    -   an OMIM (“Online Mendelian Inheritance in Man” database, Johns        Hopkins University/NCBI) public reference number for the gene,        and OMIM information such as alternative gene/protein name(s)        and/or symbol(s) in the OMIM entry.

Note that, due to the presence of alternative splice forms, multipletranscript/protein entries may be provided for a single gene entry inTable 1; i.e., for a single Gene Number, multiple entries may beprovided in series that differ in their transcript/protein informationand sequences.

Following the gene/transcript/protein information is a transcriptcontext sequence (Table 1), or a genomic context sequence (Table 2), foreach SNP within that gene.

After the last gene sequence, Table 2 may include additional genomicsequences of intergenic regions (in such instances, these sequences areidentified as “Intergenic region:” followed by a numericalidentification number), as well as SNP context sequences and other SNPinformation for any SNPs that lie within each intergenic region (suchSNPs are identified as “INTERGENIC” for SNP type).

Note that the transcript, protein, and transcript-based SNP contextsequences are all provided in the Sequence Listing. The transcript-basedSNP context sequences are provided in both Table 1 and also in theSequence Listing. The genomic and genomic-based SNP context sequencesare provided in the Sequence Listing. The genomic-based SNP contextsequences are provided in both Table 2 and in the Sequence Listing. SEQID NOs are indicated in Table 1 for the transcript-based contextsequences (SEQ ID NOS:103-176); SEQ ID NOs are indicated in Table 2 forthe genomic-based context sequences (SEQ ID NOS:623-3661).

The SNP information includes:

-   -   Context sequence (taken from the transcript sequence in Table 1,        the genomic sequence in Table 2) with the SNP represented by its        IUB code, including 100 bp upstream (5′) of the SNP position        plus 100 bp downstream (3′) of the SNP position (the        transcript-based SNP context sequences in Table 1 are provided        in the Sequence Listing as SEQ ID NOS:103-176; the genomic-based        SNP context sequences in Table 2 are provided in the Sequence        Listing as SEQ ID NOS:623-3661).    -   Celera hCV internal identification number for the SNP (in some        instances, an “hDV” number is given instead of an “hCV” number).    -   The corresponding public identification number for the SNP, the        rs number.    -   “SNP Chromosome Position” indicates the nucleotide position of        the SNP along the entire sequence of the chromosome as provided        in NCBI Genome Build 36.    -   SNP position (nucleotide position of the SNP within the given        transcript sequence (Table 1) or within the given genomic        sequence (Table 2)).    -   “Related Interrogated SNP” is the interrogated SNP with which        the listed SNP is in LD at the given value of Power.    -   SNP source (may include any combination of one or more of the        following five codes, depending on which internal sequencing        projects and/or public databases the SNP has been observed in:        “Applera”=SNP observed during the re-sequencing of genes and        regulatory regions of 39 individuals, “Celera”=SNP observed        during shotgun sequencing and assembly of the Celera human        genome sequence, “Celera Diagnostics”=SNP observed during        re-sequencing of nucleic acid samples from individuals who have        a disease, “dbSNP”=SNP observed in the dbSNP public database,        “HGBASE”=SNP observed in the HGBASE public database, “HGMD”=SNP        observed in the Human Gene Mutation Database (HGMD) public        database, “HapMap”=SNP observed in the International HapMap        Project public database, “CSNP”=SNP observed in an internal        Applied Biosystems (Foster City, CA) database of coding SNPS        (cSNPs).

Note that multiple “Applera” source entries for a single SNP indicatethat the same SNP was covered by multiple overlapping amplificationproducts and the re-sequencing results (e.g., observed allele counts)from each of these amplification products is being provided.

-   -   Population/allele/allele count information in the format of        [population1(first_allele,countlsecond_allele,count)population2(first_allele,countlsecond_allele,count)        total (first_allele,total count|second_allele,total count)]. The        information in this field includes populations/ethnic groups in        which particular SNP alleles have been observed        (“cau”=Caucasian, “his”=Hispanic, “chn”=Chinese, and        “afr”=African-American, “jpn”=Japanese, “ind”=Indian,        “mex”=Mexican, “ain”=“American Indian, “cra”=Celera donor,        “no_pop”=no population information available), identified SNP        alleles, and observed allele counts (within each population        group and total allele counts), where available [“-” in the        allele field represents a deletion allele of an        insertion/deletion (“indel”) polymorphism (in which case the        corresponding insertion allele, which may be comprised of one or        more nucleotides, is indicated in the allele field on the        opposite side of the “|”); “-” in the count field indicates that        allele count information is not available]. For certain SNPs        from the public dbSNP database, population/ethnic information is        indicated as follows (this population information is publicly        available in dbSNP): “HISP1”=human individual DNA (anonymized        samples) from 23 individuals of self-described HISPANIC        heritage; “PAC1”=human individual DNA (anonymized samples) from        24 individuals of self-described PACIFIC RIM heritage;        “CAUC1”=human individual DNA (anonymized samples) from 31        individuals of self-described CAUCASIAN heritage; “AFR1”=human        individual DNA (anonymized samples) from 24 individuals of        self-described AFRICAN/AFRICAN AMERICAN heritage; “P1”=human        individual DNA (anonymized samples) from 102 individuals of        self-described heritage; “PA130299515”; “SC_12_A”=SANGER 12 DNAs        of Asian origin from Corielle cell repositories, 6 of which are        male and 6 female; “SC_12_C”=SANGER 12 DNAs of Caucasian origin        from Corielle cell repositories from the CEPH/UTAH library, six        male and six female; “SC_12_AA”=SANGER 12 DNAs of        African-American origin from Corielle cell repositories 6 of        which are male and 6 female; “SC_95_C”=SANGER 95 DNAs of        Caucasian origin from Corielle cell repositories from the        CEPH/UTAH library; and “SC_12_CA”=Caucasians—12 DNAs from        Corielle cell repositories that are from the CEPH/UTAH library,        six male and six female.

Note that for SNPs of “Applera” SNP source, genes/regulatory regions of39 individuals (20 Caucasians and 19 African Americans) werere-sequenced and, since each SNP position is represented by twochromosomes in each individual (with the exception of SNPs on X and Ychromosomes in males, for which each SNP position is represented by asingle chromosome), up to 78 chromosomes were genotyped for each SNPposition. Thus, the sum of the African-American (“afr”) allele counts isup to 38, the sum of the Caucasian allele counts (“cau”) is up to 40,and the total sum of all allele counts is up to 78.

Note that semicolons separate population/allele/count informationcorresponding to each indicated SNP source; i.e., if four SNP sourcesare indicated, such as “Celera,” “dbSNP,” “HGBASE,” and “HGMD,” thenpopulation/allele/count information is provided in four groups which areseparated by semicolons and listed in the same order as the listing ofSNP sources, with each population/allele/count information groupcorresponding to the respective SNP source based on order; thus, in thisexample, the first population/allele/count information group wouldcorrespond to the first listed SNP source (Celera) and the thirdpopulation/allele/count information group separated by semicolons wouldcorrespond to the third listed SNP source (HGBASE); ifpopulation/allele/count information is not available for any particularSNP source, then a pair of semicolons is still inserted as aplace-holder in order to maintain correspondence between the list of SNPsources and the corresponding listing of population/allele/countinformation.

-   -   SNP type (e.g., location within gene/transcript and/or predicted        functional effect) [“MISSENSE MUTATION”=SNP causes a change in        the encoded amino acid (i.e., a non-synonymous coding SNP);        “SILENT MUTATION”=SNP does not cause a change in the encoded        amino acid (i.e., a synonymous coding SNP); “STOP CODON        MUTATION”=SNP is located in a stop codon; “NONSENSE        MUTATION”=SNP creates or destroys a stop codon; “UTR 5”=SNP is        located in a 5′ UTR of a transcript; “UTR 3”=SNP is located in a        3′ UTR of a transcript; “PUTATIVE UTR 5”=SNP is located in a        putative 5′ UTR; “PUTATIVE UTR 3”=SNP is located in a putative        3′ UTR; “DONOR SPLICE SITE”=SNP is located in a donor splice        site (5′ intron boundary); “ACCEPTOR SPLICE SITE”=SNP is located        in an acceptor splice site (3′ intron boundary); “CODING        REGION”=SNP is located in a protein-coding region of the        transcript; “EXON”=SNP is located in an exon; “INTRON”=SNP is        located in an intron; “hmCS”=SNP is located in a human-mouse        conserved segment; “TFBS”=SNP is located in a transcription        factor binding site; “UNKNOWN”=SNP type is not defined;        “INTERGENIC”=SNP is intergenic, i.e., outside of any gene        boundary].    -   Protein coding information (Table 1 only), where relevant, in        the format of [protein SEQ ID NO, amino acid position, (amino        acid-1, codon1) (amino acid-2, codon2)]. The information in this        field includes SEQ ID NO of the encoded protein sequence,        position of the amino acid residue within the protein identified        by the SEQ ID NO that is encoded by the codon containing the        SNP, amino acids (represented by one-letter amino acid codes)        that are encoded by the alternative SNP alleles (in the case of        stop codons, “X” is used for the one-letter amino acid code),        and alternative codons containing the alternative SNP        nucleotides which encode the amino acid residues (thus, for        example, for missense mutation-type SNPs, at least two different        amino acids and at least two different codons are generally        indicated; for silent mutation-type SNPs, one amino acid and at        least two different codons are generally indicated, etc.). In        instances where the SNP is located outside of a protein-coding        region (e.g., in a UTR region), “None” is indicated following        the protein SEQ ID NO.

Description of Table 3

Table 3 provides a list of linkage disequilibrium (LD) SNPs that arerelated to and derived from certain interrogated SNPs. The interrogatedSNPs, which are those SNPs provided in Tables 4-22, are statisticallysignificantly associated with, for example, response to statin treatmentfor reducing CVD/CHD risk, as described and shown herein. The LD SNPsprovided in Table 3 all have an r² value at or above 0.9 (which was setas the Threshold r² value), and are provided as examples of SNPs whichcan also be used as markers for, for example, response to statintreatment for reducing risk of CVD (especially CHD, such as MI and othercoronary events, as well as cerebrovascular events such as stroke) basedon their being in high LD with an interrogated statinresponse-associated SNP.

In Table 3, the columns labeled “Interrogated SNP” presents eachinterrogated SNP as identified by its unique hCV and rs identificationnumber. The columns labeled “LD SNP” presents the hCV and rs numbers ofthe LD SNPs that are derived from their corresponding interrogated SNPs.The column labeled “Threshold r²” presents the minimum value of r² thatan LD SNP must meet in reference to an interrogated SNP in order to beincluded in Table 3 (the Threshold r² value is set at 0.9 for all SNPsin Table 3). The column labeled “r²” presents the actual r² value of theLD SNP in reference to the interrogated SNP to which it is related(since the Threshold r² value is set at 0.9, all SNPs in Table 3 willhave an r² value at or above 0.9). The criteria for selecting the LDSNPs provided in Table 3 are further described in Example 4 below.

Sequences, SNP information, and associated gene/transcript/proteininformation for each of the LD SNPs listed in Table 3 is provided inTables 1-2.

Description of Tables 4-22

Tables 4-22 provide the results of analyses for SNPs disclosed in Tables1 and 2 (SNPs can be cross-referenced between all the tables hereinbased on their hCV and/or rs identification numbers). The results shownin Tables 4-22 provide support for the association of these SNPs with,for example, response to statin treatment for reducing the risk of CVD,particularly CHD (e.g., MI) and stroke.

Tables 4-8

The analyses in Tables 4-8 are further described in Example 1 below.

Cohort and case-only study designs were used to identify SNPs associatedwith response to statin treatment in sample sets from the CARE, WOSCOPS,and PROVE-IT trials (these sample sets, with corresponding references,are described in Example 1 below). Specifically, analyses were carriedout using the entire cohorts (individuals with and without incident CHDor CVD events) or cases only (only individuals with an incident CHD orCVD event) from these three sample sets (individually, as well as incombined meta-analyses) to identify SNPs associated with a reduction inthe risk of CHD or CVD (CVD includes CHD and stroke) in response tostatin treatment, and the results of these analyses are provided inTable 4-7 (Table 8 provides the degree of LD (r²) between pairs of SNPslisted in Tables 5 and 7).

Tables 4-7 provides SNPs that had a synergy index (odds ratio) with Pvalue lower than 10⁻⁴ in a meta-analysis of CARE and WOSCOPS combined(Table 4-5) or in a meta-analysis of CARE, WOSCOPS, and PROVE-ITcombined (Table 6-7), in any genetic model (dominant, recessive, oradditive) in either the CHD or CVD endpoint (the CHD or CVD endpoint isindicated in the last column, labeled “Endpoint”, of Tables 4-7, and thegenetic model is indicated in the next to last column, labeled “Model”,of Tables 4-7). For each analysis, Tables 4-7 indicate whether the datacomes from case-only analysis (“CaseOnly” in the “Source” column) orfrom analysis of the entire cohort (“cohort” in the “Source” column).Whenever cohort data was available, it was used in the meta-analysis.

Tables 4-5 provide meta-analyses of CARE and WOSCOPS combined (2^(nd)section of each table) for two endpoints (CHD and CVD) and three geneticmodels (dominant, recessive, and additive), as well as logisticregression analyses of CARE (3^(rd) section of each table) and WOSCOPS(4^(th) section of each table) individually.

Tables 6-7 provide meta-analyses of CARE, WOSCOPS, and PROVE-IT combined(2^(nd) section of each table) for two endpoints (CHD and CVD) and threegenetic models (dominant, recessive, and additive), as well as logisticregression analyses of CARE (3^(rd) section of each table), WOSCOPS(4^(th) section of each table), and PROVE-IT (5^(th) section of eachtable) individually. For PROVE-IT, there was only one endpoint (thecomposite primary endpoint of the original PROVE-IT study, whichincludes some stroke cases), and this endpoint was used in meta-analysisof both CHD and CVD.

Tables 5 and 7 provide analyses of certain LD SNPs in CARE and WOSCOPS(Table 5) and in CARE, WOSCOPS, and PROVE-IT (Table 7). For some SNPs,case-only data was available for a first SNP while cohort data wasavailable for a SNP in LD with the first SNP (LD SNP), which occurredwhen a working kPCR assay could not be made for the first SNP. For theseSNPs, the data for case-only analysis and the available data for thecohort is reported. The meta-analysis was performed using the cohortdata when available. These SNPs are listed in Tables 5 and 7, with thetwo SNPs in LD listed one below the other, and the degree of LD betweeneach of these pairs of SNPs is provided in Table 8.

Notations in Tables 4-7 are as follows:

In Tables 4-7, “allele A1” may be interchangeably referred to as the“non-reference allele” (“non-ref”), and “allele A2” may beinterchangeably referred to as the “reference allele” (“ref”). The OR'sthat are indicated in Tables 4-7 correspond to the indicated“non-reference allele” (“allele A1”). Thus, if OR<1, the “non-referenceallele” (“allele A1”) is associated with reduction of CVD/CHD risk bystatin treatment, whereas if OR>1, the other alternative allele at theSNP (the “reference allele” or “allele A2”) is associated with reductionof CVD/CHD risk by statin treatment.

The counts are indicated in the following format: allele A1homozygotes/heterozygotes/allele A2 homozygotes. These counts indicatethe number of individuals in the pravastatin (“Prava”), placebo, oratorvastatin (“Atorva”) arms of the CARE, WOSCOPS, or PROVE-IT trials(as indicated) who have the corresponding genotypes.

In Tables 4-7, “P value” indicates the p-value, “OR” indicates the oddsratio (synergy index), “OR L95” and “OR U95” indicates the lower andupper (respectively) 95% confidence interval for the odds ratio, and“Source” indicates whether the data comes from case-only analysis(“CaseOnly”) or from analysis of the entire cohort (“cohort”).

Tables 9-18

Tables 9-18 provide additional SNPs associated with response to statintreatment for reducing CVD/CHD risk. Tables 9-18 differ from Tables 4-8in that Tables 9-18 include SNPs analyzed by imputation as well as bygenotyping, whereas all of the SNPs in Tables 4-8 were analyzed bygenotyping. Imputation involves imputing the allele/genotype present ata SNP for each individual in the sample set (CARE, WOSCOPS, andPROVE-IT) rather than directly genotyping the SNP in a sample from theindividual. The column labeled “Source” in each of Tables 9-18 indicateswhether the data presented for each SNP was derived from imputation orfrom genotyping.

Specifically, analyses were carried out using the same three sample setsas in Tables 4-8 (CARE, WOSCOPS, and PROVE-IT) to identify (by bothgenotyping and imputation) additional SNPs beyond those provided inTables 4-8 that are also associated with a reduction in the risk of CHDor CVD. Tables 9-18 provide results of analyses of statin response forthe same two endpoints as in Tables 4-8 (CHD in Tables 9-13, and CVD inTables 14-18) and four genetic models (dominant, recessive, additive,and genotypic 2df).

Tables 9-18 provide genotyped and imputed SNPs for which the p-value fora random effect was lower than 10⁻⁴ for either the meta-analysis of CAREand WOSCOPS combined or the meta-analysis of CARE, WOSCOPS, and PROVE-ITcombined, for either the CHD or CVD endpoint, and for any genetic model(dominant, recessive, additive, or genotypic). Association interactionbetween statin response and either the CHD or CVD phenotype wasperformed.

Tables 9-13 have CHD as an endpoint, whereas Tables 14-18 have CVD as anendpoint (CVD includes CHD and stroke).

Tables 9 and 14 provide results of logistic regression analysis of theCARE sample set by direct genotyping and by imputing genotypes.

Tables 10 and 15 provide results of logistic regression analysis of theWOSCOPS sample set by direct genotyping and by imputing genotypes.

Tables 11 and 16 provide results of logistic regression analysis of thePROVE-IT sample set by direct genotyping and by imputing genotypes.

Tables 12 and 17 provide results of meta-analysis of the CARE andWOSCOPS sample sets combined by direct genotyping and by imputinggenotypes.

Tables 13 and 18 provide results of meta-analysis of the CARE, WOSCOPS,and PROVE-IT sample sets combined by direct genotyping and by imputinggenotypes.

Notations in Tables 9-11 and 14-16 (for the analysis of CARE, WOSCOPS,and PROVE-IT sample sets individually) are as follows:

“SOURCE” indicates whether each SNP was genotyped (“Genotyped”) orimputed (“Imputed”).

“ALLELE” indicates the allele for which the given data (such as the OR)correspond to, which is also referred to herein as allele “A1” (and theother alternative allele at each SNP, which is not shown in Tables 9-11and 14-16, but is shown in Tables 1-2 for each SNP, is referred to asallele “A2”).

“MODEL” indicates whether the model was additive (“ADD”), recessive(“REC”), dominant (“DOM”), or genotypic 2df (“GEN”).

“NMISS” indicates the number of genotypes present in the analysis (thenumber of non-missing genotypes).

“OR” indicates the odds ratio (synergy index (SI)). If the odds ratio isless than one for the indicated allele (i.e., allele A1) then thisindicates that this allele is associated with statin response (benefitfrom statin treatment), i.e., fewer CVD or CHD events (e.g., MI) wereobserved in individuals with this allele in the pravastatin arm of CAREor WOSCOPS or the atorvastatin arm of PROVE-IT, relative to individualswith this allele in the placebo arm of CARE or WOSCOPS or thepravastatin arm of PROVE-IT. If the odds ratio is greater than one forthe indicated allele, then this indicates that the other alternativeallele at the SNP (the allele which is not shown in Tables 9-11 and14-16, but is indicated in Tables 1-2, i.e., allele A2), is associatedwith statin response (benefit from statin treatment).

“SE” indicates standard error of the natural log of the synergy index(the synergy index is the odds ratio, labeled “OR”).

“L95” and “U95” indicates the lower and upper (respectively) 95%confidence interval for the odds ratio.

“STAT” is the test statistic used in evaluating the significance of anassociation in logistic regression analysis. The statistic is equal tothe natural log of the synergy index divided by its standard error andfollows a Gaussian distribution under the null hypothesis that thesynergy index is equal to one.

“P” indicates the p-value (corresponding to a statistical test ofwhether the synergy index is equal to one), and “HW_PVALUE” indicatesthe p-value corresponding to a statistical test of whether thedistribution of genotypes among subjects in the study agrees with thedistribution expected according to Hardy-Weinberg equilibrium.

“ALLELE_FREQ” indicates the allele frequency of the given allele in theanalyzed sample set (CARE in Tables 9 and 14; WOSCOPS in Tables 10 and15; or PROVE-IT in Tables 11 and 16).

“PRAVA_ALLELE_FREQ” or “ATORVA_ALLELE_FREQ” indicates the allelefrequency of the given allele in the pravastatin or atorvastatin-treatedarms (respectively) of the CARE, WOSCOPS, or PROVE-IT trials.

“PRAVA_A1_HZ_COUNT”, “PRAVA_HET_COUNT”, and “PRAVA_A2_HZ_COUNT” (or, inTables 11 and 16, “ATORVA_A1_HZ_COUNT”, “ATORVA_HET_COUNT”, and“ATORVA_A2_HZ_COUNT”) indicate the number of homozygotes of the allelethat is indicated in the table (allele A1), the number of heterozygotes,and the number of homozygotes of the other alternative allele (alleleA2) at the SNP, respectively, in the pravastatin arm of the CARE trial(in Tables 9 and 14) or the WOSCOPS trial (in Tables 10 and 15), or inthe atorvastatin arm of the PROVE-IT trial (in Tables 11 and 16, inwhich the column headings labeled “atorvastatin” (“atorva”) areanalogous to the column headings labeled “pravastatin” (“prava”) inTables 9-10 and 14-15).

“PLACEBO_A1_HZ_COUNT”, “PLACEBO_HET_COUNT”, and “PLACEBO_A2_HZ_COUNT”(or, in Tables 11 and 16, “PRAVA_A1_HZ_COUNT”, “PRAVA_HET_COUNT”, and“PRAVA_A2_HZ_COUNT”) indicate the number of homozygotes of the allelethat is indicated in the table (allele A1), the number of heterozygotes,and the number of homozygotes of the other alternative allele (alleleA2) at the SNP, respectively, in the placebo arm of the CARE trial (inTables 9 and 14) or the WOSCOPS trial (in Tables 10 and 15), or in thepravastatin arm of the PROVE-IT trial (in Tables 11 and 16, in which thecolumn headings labeled “pravastatin” (“prava”) are analogous to thecolumn headings labeled “placebo” in Tables 9-10 and 14-15).

Notations in Tables 12-13 and 17-18 (for the meta-analysis of CARE andWOSCOPS combined, and CARE, WOSCOPS, and PROVE-IT combined) are asfollows:

“SOURCE” indicates whether each SNP was genotyped or imputed.

“ALLELE” indicates the allele for which the given data (such as the OR)correspond to, which is also referred to herein as allele “A1” (and theother alternative allele at each SNP, which is not shown in Tables 12-13and 17-18, but is shown in Tables 1-2 for each SNP, is referred to asallele “A2”).

“MODEL” indicates whether the model was additive, recessive, dominant,or genotypic.

“P” indicates the p-value, and “P(R)” (or “P.R.”) indicates the p-valuerandom effect. Both of these are p-values corresponding to a statisticaltest of whether the combined synergy index is equal to one but usedifferent assumptions to derive the p-value. “P” is calculated using afixed effects model, and “P(R)” is calculated using a random effectsmodel.

“OR” indicates the odds ratio (synergy index) calculated from a fixedeffects model, and “OR(R)” (or “OR.R.”) indicates the odds ratio(synergy index) calculated from a random effects model. If the oddsratio is less than one for the indicated allele (i.e., allele A1) thenthis indicates that this allele is associated with statin response(benefit from statin treatment), i.e., fewer CVD or CHD events (e.g.,MI) were observed in individuals with this allele in a combinedmeta-analysis of the pravastatin arms of CARE and WOSCOPS (Tables 12 and17) and the atorvastatin arm of PROVE-IT (Tables 13 and 18), relative toindividuals with this allele in the placebo arms of CARE and WOSCOPS(Tables 12 and 17) and the pravastatin arm of PROVE-IT (Tables 13 and18). If the odds ratio is greater than one for the indicated allele,then this indicates that the other alternative allele at the SNP (theallele which is not shown in Tables 12-13 and 17-18, but is indicated inTables 1-2, i.e., allele A2), is associated with statin response(benefit from statin treatment).

“Q” indicates the Cochran Q test p-value, which is a p-valuecorresponding to the statistical test of the homogeneity of the synergyindex across studies (small p-values indicate a greater degree ofheterogeneity between studies).

“I” indicates the I² heterogeneity index, which can be interpreted asthe proportion of total variation in the estimates of effect that is dueto heterogeneity between studies.

Table 19

The analysis in Table 19 is further described in Example 2 below.

Notations in Table 19 are similar to Tables 4-7. “P.R.” and “OR.R.”indicate the p-value and odds ratio (synergy index), respectively,calculated from a random effects model (rather than a fixed effectsmodel).

Table 19 shows that SNP rs11556924 (hCV31283062) in the ZC3HC1 gene isassociated with differential reduction of CHD risk by pravastatintherapy in both the CARE and WOSCOPS sample sets.

Tables 20-22

The analyses in Tables 20-22 are further described in Example 3 below.

The results shown in Tables 20-22 provide support for the association ofthese SNPs with CVD risk and/or statin response, particularly risk forstroke and/or response to statin treatment for reducing the risk ofstroke (Tables 20-21) and CHD (Table 22).

Tables 20-21 provides SNPs associated with stroke risk and/or strokestatin response (reduction in stroke risk by statin treatment) in theCARE sample set. Consistent with the CARE trial, the stroke endpoint inthe analysis for which the results are provided in Tables 20-21 includedstroke as well as transient ischemic attack (TIA).

Table 22 provides a SNP associated with CHD statin response in the CAREsample set. Table 22 shows that SNP rs873134 in the B4GALNT3 gene isassociated with response to statin treatment for reducing the risk ofCHD, particularly recurrent MI. In the analysis for which the resultsare provided in Table 22, the endpoint was recurrent MI, and theanalysis was adjusted for age, gender, hypertension, diabetes, base LDLand HDL, and whether an individual was a current smoker.

Notations in Tables 20-22 are as follows:

In Tables 20-22, certain columns are labeled “RESP”, “PLACEBO, OR “ALL”.“RESP” is for statin response as measured by comparing risk (risk forstroke, including TIA, in Tables 20-21, and risk for CHD, specificallyrecurrent MI, in Table 22) in the pravastatin-treated group with risk inthe placebo-treated group, “PLACEBO” is the placebo-treated group, and“ALL” is the combination of the placebo-treated group and thepravastatin-treated group. “RESP” is the analysis to assess statinresponse in the indicated genotype group.

“MODE” indicates whether the model was additive (“ADD”), recessive(“REC”), dominant (“DOM”), or genotypic (“GEN”).

“mAF CEU” (Table 20 only) indicates the frequency of the minor allele inHapMap for Europeans.

“GENO” indicates the genotype.

“STATIN” indicates either the pravastatin-treated (“Pravastatin”) orplacebo-treated (“Placebo”) groups (i.e., arms of the CARE trial).

“EVENTS” indicates the total number of events (stroke or TIA for Tables20-21, and recurrent MI for Table 22) in individuals with the indicatedgenotype.

“TOTAL” indicates the total number of individuals with the indicatedgenotype.

“HR” indicates the hazard ratio.

“L95” and “U95” indicates the lower and upper (respectively) 95%confidence interval for the hazard ratio.

“P” indicates the p-value, “P_INT” indicates p-interaction, “P_DF2”indicates two degrees of freedom p-value, and “HW(ALL)pExact” (Table 21only) indicates p exact for Hardy Weinberg Equilibrium for the ALLgroup.

Throughout Tables 4-22, “OR” refers to the odds ratio, “HR” refers tothe hazard ratio, and “OR L95” or “OR U95” refers to the lower and upper(respectively) 95% confidence interval for the odds ratio or hazardratio. With respect to drug response (e.g., response to a statin), ifthe OR or HR of those treated with the drug (e.g., a statin) comparedwith those treated with a placebo within a particular genotype (or witha particular allele) is less than one, this indicates that an individualwith this particular genotype or allele would benefit from the drug (anOR or HR equal to one would indicate that the drug has no effect). Incontrast, with respect to drug response, if the OR or HR is greater thanone for a particular allele, then this indicates that an individual withthe other alternative allele would benefit from the drug. As usedherein, the term “benefit” (with respect to a preventive or therapeuticdrug treatment) is defined as achieving a reduced risk for a diseasethat the drug is intended to treat or prevent (e.g., CVD such as CHD,particularly MI) by administering the drug treatment, compared with therisk for the disease in the absence of receiving the drug treatment (orreceiving a placebo in lieu of the drug treatment) for the samegenotype.

With respect to disease risk, an OR or HR that is greater than oneindicates that a given allele is a risk allele (which may also bereferred to as a susceptibility allele), whereas an OR or HR that isless than one indicates that a given allele is a non-risk allele (whichmay also be referred to as a protective allele). For a given riskallele, the other alternative allele at the SNP position (which can bederived from the information provided in Tables 1-2, for example) may beconsidered a non-risk allele. For a given non-risk allele, the otheralternative allele at the SNP position may be considered a risk allele.Thus, with respect to disease risk, if the OR or HR for a particularallele at a SNP position is greater than one, this indicates that anindividual with this particular allele has a higher risk for the diseasethan an individual who has the other allele at the SNP position. Incontrast, if the OR or HR for a particular allele is less than one, thisindicates that an individual with this particular allele has a reducedrisk for the disease compared with an individual who has the otherallele at the SNP position.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiments of the present invention provide SNPs associatedwith response to statin treament, particularly for reducing the risk ofCVD (especially CHD, such as MI and other coronary events, as well ascerebrovascular events such as stroke), and methods for their use. Thepresent invention further provides nucleic acid molecules containingthese SNPs, methods and reagents for the detection of the SNPs disclosedherein, uses of these SNPs for the development of detection reagents,and assays or kits that utilize such reagents. The statinresponse-associated SNPs disclosed herein are particularly useful forpredicting, screening for, and evaluating response to statin treatment,particularly for prevention or treatment of CVD (particularly CHD, suchas MI and other coronary events, as well as cerebrovascular events suchas stroke) using statins, in humans. The SNPs disclosed herein are alsouseful for diagnosing, prognosing, screening for, and evaluatingpredisposition to CVD, particularly CHD (such as MI) as well ascerebrovascular events such as stroke, in humans. Furthermore, such SNPsand their encoded products are useful targets for the development oftherapeutic and preventive agents.

Thus, exemplary embodiments of the present invention provide individualSNPs associated with response to statin treatments, particularly forreducing the risk of CVD (especially CHD, such as MI and other coronaryevents, as well as cerebrovascular events such as stroke), as well ascombinations of SNPs and haplotypes, polymorphic/variant transcriptsequences (SEQ ID NOS:1-51) and genomic sequences (SEQ ID NOS:177-622)containing SNPs, encoded amino acid sequences (SEQ ID NOS:52-102), andboth transcript-based SNP context sequences (SEQ ID NOS:103-176) andgenomic-based SNP context sequences (SEQ ID NOS:623-3661) (transcriptsequences, protein sequences, and transcript-based SNP context sequencesare provided in Table 1 and the Sequence Listing; genomic sequences andgenomic-based SNP context sequences are provided in Table 2 and theSequence Listing), methods of detecting these polymorphisms in a testsample, methods of determining if an individual is likely to respond toa particular treatment such as statins (particularly for treating orpreventing CVD, such as CHD or stroke), methods of determining anindividual's risk for developing CVD, methods of screening for compoundsuseful for treating CVD, compounds identified by these screeningmethods, methods of using the disclosed SNPs to select atreatment/preventive strategy or therapeutic agent, and methods oftreating or preventing CVD.

Exemplary embodiments of the present invention further provide methodsfor selecting or formulating a treatment regimen (e.g., methods fordetermining whether or not to administer statin treatment to anindividual having CVD, or who is at risk for developing CVD in thefuture, or who has previously had CVD, methods for selecting aparticular statin-based treatment regimen such as dosage and frequencyof administration of statin, or a particular form/type of statin such asa particular pharmaceutical formulation or statin compound, methods foradministering (either in addition to or instead of a statin) analternative, non-statin-based treatment, such as niacin, fibrates, orezetimibe (e.g., Zetia® or Ezetrol®), to individuals who are predictedto be unlikely to respond positively to statin treatment, etc.), andmethods for determining the likelihood of experiencing toxicity or otherundesirable side effects from statin treatment, etc. The presentinvention also provides methods for selecting individuals to whom astatin or other therapeutic will be administered based on theindividual's genotype, and methods for selecting individuals for aclinical trial of a statin or other therapeutic agent based on thegenotypes of the individuals (e.g., selecting individuals to participatein the trial who are most likely to respond positively from the statintreatment and/or excluding individuals from the trial who are unlikelyto respond positively from the statin treatment based on their SNPgenotype(s), or selecting individuals who are unlikely to respondpositively to statins based on their SNP genotype(s) to participate in aclinical trial of another type of drug that may benefit them).

Exemplary embodiments of the present invention may include novel SNPsassociated with response to statin treatment, as well as SNPs that werepreviously known in the art, but were not previously known to beassociated with response to statin treatment. Accordingly, the presentinvention may provide novel compositions and methods based on novel SNPsdisclosed herein, and may also provide novel methods of using known, butpreviously unassociated, SNPs in methods relating to, for example,methods relating to evaluating an individual's likelihood of respondingto statin treatment (particularly statin treatment, including preventivetreatment, of CVD, such as CHD or stroke), evaluating an individual'slikelihood of having or developing CVD (particularly CHD or stroke), andpredicting the likelihood of an individual experiencing a reccurrence ofCVD (e.g., experiencing recurrent MI). In Tables 1 and 2, known SNPs areidentified based on the public database in which they have beenobserved, which is indicated as one or more of the following SNP types:“dbSNP”=SNP observed in dbSNP, “HGBASE”=SNP observed in HGBASE, and“HGMD”=SNP observed in the Human Gene Mutation Database (HGMD).

Particular alleles of the SNPs disclosed herein can be associated witheither an increased likelihood of responding to statin treatment(particularly for reducing the risk of CVD, such as CHD (e.g., MI) orstroke) or increased risk of developing CVD (e.g., CHD or stroke), or adecreased likelihood of responding to statin treatment or a decreasedrisk of developing CVD. Thus, whereas certain SNPs (or their encodedproducts) can be assayed to determine whether an individual possesses aSNP allele that is indicative of an increased likelihood of respondingto statin treatment or an increased risk of developing CVD, other SNPs(or their encoded products) can be assayed to determine whether anindividual possesses a SNP allele that is indicative of a decreasedlikelihood of responding to statin treatment or a decreased risk ofdeveloping CVD. Similarly, particular alleles of the SNPs disclosedherein can be associated with either an increased or decreasedlikelihood of having a reccurrence of CVD (e.g., recurrent MI), etc. Theterm “altered” may be used herein to encompass either of these twopossibilities (e.g., either an increased or a decreasedlikelihood/risk).

SNP alleles that are associated with increased response to statintreatment for reducing CVD risk (benefit from statin treatment) may bereferred to as “response” alleles, and SNP alleles that are associatedwith a lack of response to statin treatment may be referred to as“non-response” alleles. SNP alleles that are associated with anincreased risk of having or developing CVD may be referred to as “risk”or “susceptibility” alleles, and SNP alleles that are associated with adecreased risk of having or developing CVD may be referred to as“non-risk” or “protective” alleles.

In certain embodiments, the presence of a statin response alleledisclosed herein in Tables 4-22 (an allele associated with increasedresponse to statin treatment for reducing CVD or CHD risk) is detectedand indicates that an individual has an increased risk for developingCVD, such as CHD (e.g., MI) or stroke. In these embodiments, in whichthe same allele is associated with both increased risk for developingCVD and increased response to statin treatment (i.e., the same allele isboth a risk and a response allele), this increased risk for developingCVD can be reduced by administering statin treatment to an individualhaving the allele.

Those skilled in the art will readily recognize that nucleic acidmolecules may be double-stranded molecules and that reference to aparticular site on one strand refers, as well, to the corresponding siteon a complementary strand. In defining a SNP position, SNP allele, ornucleotide sequence, reference to an adenine, a thymine (uridine), acytosine, or a guanine at a particular site on one strand of a nucleicacid molecule also defines the thymine (uridine), adenine, guanine, orcytosine (respectively) at the corresponding site on a complementarystrand of the nucleic acid molecule. Thus, reference may be made toeither strand in order to refer to a particular SNP position, SNPallele, or nucleotide sequence. Probes and primers, may be designed tohybridize to either strand and SNP genotyping methods disclosed hereinmay generally target either strand. Throughout the specification, inidentifying a SNP position, reference is generally made to theprotein-encoding strand, only for the purpose of convenience.

References to variant peptides, polypeptides, or proteins of the presentinvention include peptides, polypeptides, proteins, or fragmentsthereof, that contain at least one amino acid residue that differs fromthe corresponding amino acid sequence of the art-knownpeptide/polypeptide/protein (the art-known protein may beinterchangeably referred to as the “wild-type,” “reference,” or “normal”protein). Such variant peptides/polypeptides/proteins can result from acodon change caused by a nonsynonymous nucleotide substitution at aprotein-coding SNP position (i.e., a missense mutation) disclosed by thepresent invention. Variant peptides/polypeptides/proteins of the presentinvention can also result from a nonsense mutation (i.e., a SNP thatcreates a premature stop codon, a SNP that generates a read-throughmutation by abolishing a stop codon), or due to any SNP disclosed by thepresent invention that otherwise alters the structure, function,activity, or expression of a protein, such as a SNP in a regulatoryregion (e.g. a promoter or enhancer) or a SNP that leads to alternativeor defective splicing, such as a SNP in an intron or a SNP at anexon/intron boundary. As used herein, the terms “polypeptide,”“peptide,” and “protein” are used interchangeably.

As used herein, an “allele” may refer to a nucleotide at a SNP position(wherein at least two alternative nucleotides exist in the population atthe SNP position, in accordance with the inherent definition of a SNP)or may refer to an amino acid residue that is encoded by the codon whichcontains the SNP position (where the alternative nucleotides that arepresent in the population at the SNP position form alternative codonsthat encode different amino acid residues). An “allele” may also bereferred to herein as a “variant”. Also, an amino acid residue that isencoded by a codon containing a particular SNP may simply be referred toas being encoded by the SNP.

A phrase such as “as represented by”, “as shown by”, “as symbolized by”,or “as designated by” may be used herein to refer to a SNP within asequence (e.g., a polynucleotide context sequence surrounding a SNP),such as in the context of “a polymorphism as represented by position 101of SEQ ID NO:X or its complement”. Typically, the sequence surrounding aSNP may be recited when referring to a SNP, however the sequence is notintended as a structural limitation beyond the specific SNP positionitself. Rather, the sequence is recited merely as a way of referring tothe SNP (in this example, “SEQ ID NO:X or its complement” is recited inorder to refer to the SNP located at position 101 of SEQ ID NO:X, butSEQ ID NO:X or its complement is not intended as a structural limitationbeyond the specific SNP position itself). In other words, it isrecognized that the context sequence of SEQ ID NO:X in this example maycontain one or more polymorphic nucleotide positions outside of position101 and therefore an exact match over the full-length of SEQ ID NO:X isirrelevant since SEQ ID NO:X is only meant to provide context forreferring to the SNP at position 101 of SEQ ID NO:X. Likewise, thelength of the context sequence is also irrelevant (100 nucleotides oneach side of a SNP position has been arbitrarily used in the presentapplication as the length for context sequences merely for convenienceand because 201 nucleotides of total length is expected to providesufficient uniqueness to unambiguously identify a given nucleotidesequence). Thus, since a SNP is a variation at a single nucleotideposition, it is customary to refer to context sequence (e.g., SEQ IDNO:X in this example) surrounding a particular SNP position in order touniquely identify and refer to the SNP. Alternatively, a SNP can bereferred to by a unique identification number such as a public “rs”identification number or an internal “hCV” identification number, suchas provided herein for each SNP (e.g., in Tables 1-2). For example, inthe instant application, “rs11556924”, “hCV31283062”, and “position 101of SEQ ID NO:1074” all refer to the same SNP.

As used herein, the term “benefit” (with respect to a preventive ortherapeutic drug treatment, such as statin treatment) is defined asachieving a reduced risk for a disease that the drug is intended totreat or prevent (e.g., CVD such as CHD (particularly MI) or stroke) byadministrating the drug treatment, compared with the risk for thedisease in the absence of receiving the drug treatment (or receiving aplacebo in lieu of the drug treatment) for the same genotype. The term“benefit” may be used herein interchangeably with terms such as “respondpositively” or “positively respond”.

As used herein, the terms “drug” and “therapeutic agent” are usedinterchangeably, and may include, but are not limited to, small moleculecompounds, biologics (e.g., antibodies, proteins, protein fragments,fusion proteins, glycoproteins, etc.), nucleic acid agents (e.g.,antisense, RNAi/siRNA, and microRNA molecules, etc.), vaccines, etc.,which may be used for therapeutic and/or preventive treatment of adisease (e.g., CVD such as CHD or stroke).

Examples of statins (also known as HMG-CoA reductase inhibitors)include, but are not limited to, atorvastatin (Lipitor®), rosuvastatin(Crestor®), pravastatin (Pravachol®), simvastatin (Zocor®), fluvastatin(Lescol®), and lovastatin (Mevacor®), as well as combination therapiesthat include a statin such as simvastatin+ezetimibe (Vytorin®),lovastatin+niacin (Advicor®), atorvastatin+amlodipine besylate(Caduet®), and simvastatin+niacin (Simcor®).

Certain exemplary embodiments of the invention provide the followingcompositions and uses: (1) a reagent (such as an allele-specific probeor primer, or any other oligonucleotide or other reagent suitable fordetecting a polymorphism disclosed herein, which can include detectionof any allele of the polymorphism) for use as a diagnostic or predictiveagent for determining statin response, particularly for reducing therisk of CVD such as CHD (e.g., MI) or stroke (and/or for determiningrisk for developing CVD); (2) a kit, device, array, or assay componentthat includes or is coupled with the reagent of (1) above for use indetermining statin response, particularly for reducing the risk of CVD(and/or for determining risk for developing CVD); (3) the use of thereagent of (1) above for the manufacture of a kit, device, array, orassay component for determining statin response, particularly forreducing the risk of CVD (and/or for determining risk for CVD); and (4)the use of a polymorphism disclosed herein for the manufacture of areagent for use as a diagnostic or predictive agent for determiningstatin response, particularly for reducing the risk of CVD (and/or fordetermining risk for developing CVD).

The various methods described herein, such as correlating the presenceor absence of a polymorphism with the predicted response of anindividual to a drug such as a statin, particularly for reducing therisk for CVD such as CHD (e.g., MI) or stroke (and/or correlating thepresence or absence of a polymorphism with an altered (e.g., increasedor decreased) risk (or no altered risk) for developing CVD), can becarried out by automated methods such as by using a computer (or otherapparatus/devices such as biomedical devices, laboratoryinstrumentation, or other apparatus/devices having a computer processor)programmed to carry out any of the methods described herein. Forexample, computer software (which may be interchangeably referred toherein as a computer program) can perform the step of correlating thepresence or absence of a polymorphism in an individual with an altered(e.g., increased or decreased) response (or no altered response) tostatin treatment, particularly for reducing the risk for CVD such as CHD(e.g., MI) or stroke. Accordingly, certain embodiments of the inventionprovide a computer (or other apparatus/device) programmed to carry outany of the methods described herein.

Reports, Programmed Computers, Business Methods, and Systems

The results of a test (e.g., an individual's predicted responsiveness tostatin treatment for reducing CVD risk, or an individual's risk fordeveloping CVD, based on assaying one or more SNPs disclosed herein,and/or an individual's allele(s)/genotype at one or more SNPs disclosedherein, etc.), and/or any other information pertaining to a test, may bereferred to herein as a “report”. A tangible report can optionally begenerated as part of a testing process (which may be interchangeablyreferred to herein as “reporting”, or as “providing” a report,“producing” a report, or “generating” a report).

Examples of tangible reports may include, but are not limited to,reports in paper (such as computer-generated printouts of test results)or equivalent formats and reports stored on computer readable medium(such as a CD, USB flash drive or other removable storage device,computer hard drive, or computer network server, etc.). Reports,particularly those stored on computer readable medium, can be part of adatabase, which may optionally be accessible via the internet (such as adatabase of patient records or genetic information stored on a computernetwork server, which may be a “secure database” that has securityfeatures that limit access to the report, such as to allow only thepatient and the patient's medical practioners to view the report whilepreventing other unauthorized individuals from viewing the report, forexample). In addition to, or as an alternative to, generating a tangiblereport, reports can also be displayed on a computer screen (or thedisplay of another electronic device or instrument).

A report can include, for example, an individual's predictedresponsiveness to statin treatment (e.g., whether the individual willbenefit from statin treatment by having their risk for CVD, particularlyCHD (e.g., MI) or stroke, reduced), or may just include theallele(s)/genotype that an individual carries at one or more SNPsdisclosed herein, which may optionally be linked to informationregarding the significance of having the allele(s)/genotype at the SNP(for example, a report on computer readable medium such as a networkserver may include hyperlink(s) to one or more journal publications orwebsites that describe the medical/biological implications, such asstatin response and/or CVD risk, for individuals having a certainallele/genotype at the SNP). Thus, for example, the report can includedrug responsiveness, disease risk, and/or other medical/biologicalsignificance, as well as optionally also including the allele/genotypeinformation, or the report may just include allele/genotype informationwithout including drug responsiveness, disease risk, or othermedical/biological significance (such that an individual viewing thereport can use the allele/genotype information to determine theassociated drug response, disease risk, or other medical/biologicalsignificance from a source outside of the report itself, such as from amedical practioner, publication, website, etc., which may optionally belinked to the report such as by a hyperlink).

A report can further be “transmitted” or “communicated” (these terms maybe used herein interchangeably), such as to the individual who wastested, a medical practitioner (e.g., a doctor, nurse, clinicallaboratory practitioner, genetic counselor, etc.), a healthcareorganization, a clinical laboratory, and/or any other party or requesterintended to view or possess the report. The act of “transmitting” or“communicating” a report can be by any means known in the art, based onthe format of the report. Furthermore, “transmitting” or “communicating”a report can include delivering/sending a report (“pushing”) and/orretrieving (“pulling”) a report. For example, reports can betransmitted/communicated by various means, including being physicallytransferred between parties (such as for reports in paper format) suchas by being physically delivered from one party to another, or by beingtransmitted electronically or in signal form (e.g., via e-mail or overthe internet, by facsimile, and/or by any wired or wirelesscommunication methods known in the art) such as by being retrieved froma database stored on a computer network server, etc.

In certain exemplary embodiments, the invention provides computers (orother apparatus/devices such as biomedical devices or laboratoryinstrumentation) programmed to carry out the methods described herein.For example, in certain embodiments, the invention provides a computerprogrammed to receive (i.e., as input) the identity (e.g., the allele(s)or genotype at a SNP) of one or more SNPs disclosed herein and provide(i.e., as output) the predicted drug responsiveness or disease risk(e.g., an individual's predicted statin responsiveness or risk fordeveloping CVD) or other result based on the identity of the SNP(s).Such output (e.g., communication of disease risk, disease diagnosis orprognosis, drug responsiveness, etc.) may be, for example, in the formof a report on computer readable medium, printed in paper form, and/ordisplayed on a computer screen or other display.

In various exemplary embodiments, the invention further provides methodsof doing business (with respect to methods of doing business, the terms“individual” and “customer” are used herein interchangeably). Forexample, exemplary methods of doing business can comprise assaying oneor more SNPs disclosed herein and providing a report that includes, forexample, a customer's predicted response to statin treatment (e.g., forreducing their risk for CVD, particularly CHD (such as MI) or stroke) ortheir risk for developing CVD (based on which allele(s)/genotype ispresent at the assayed SNP(s)) and/or that includes theallele(s)/genotype at the assayed SNP(s) which may optionally be linkedto information (e.g., journal publications, websites, etc.) pertainingto disease risk or other biological/medical significance such as bymeans of a hyperlink (the report may be provided, for example, on acomputer network server or other computer readable medium that isinternet-accessible, and the report may be included in a secure databasethat allows the customer to access their report while preventing otherunauthorized individuals from viewing the report), and optionallytransmitting the report. Customers (or another party who is associatedwith the customer, such as the customer's doctor, for example) canrequest/order (e.g., purchase) the test online via the internet (or byphone, mail order, at an outlet/store, etc.), for example, and a kit canbe sent/delivered (or otherwise provided) to the customer (or anotherparty on behalf of the customer, such as the customer's doctor, forexample) for collection of a biological sample from the customer (e.g.,a buccal swab for collecting buccal cells), and the customer (or a partywho collects the customer's biological sample) can submit theirbiological samples for assaying (e.g., to a laboratory or partyassociated with the laboratory such as a party that accepts the customersamples on behalf of the laboratory, a party for whom the laboratory isunder the control of (e.g., the laboratory carries out the assays byrequest of the party or under a contract with the party, for example),and/or a party that receives at least a portion of the customer'spayment for the test). The report (e.g., results of the assay including,for example, the customer's disease risk and/or allele(s)/genotype atthe assayed SNP(s)) may be provided to the customer by, for example, thelaboratory that assays the SNP(s) or a party associated with thelaboratory (e.g., a party that receives at least a portion of thecustomer's payment for the assay, or a party that requests thelaboratory to carry out the assays or that contracts with the laboratoryfor the assays to be carried out) or a doctor or other medicalpractitioner who is associated with (e.g., employed by or having aconsulting or contracting arrangement with) the laboratory or with aparty associated with the laboratory, or the report may be provided to athird party (e.g., a doctor, genetic counselor, hospital, etc.) whichoptionally provides the report to the customer. In further embodiments,the customer may be a doctor or other medical practitioner, or ahospital, laboratory, medical insurance organization, or other medicalorganization that requests/orders (e.g., purchases) tests for thepurposes of having other individuals (e.g., their patients or customers)assayed for one or more SNPs disclosed herein and optionally obtaining areport of the assay results.

In certain exemplary methods of doing business, a kit for collecting abiological sample (e.g., a buccal swab for collecting buccal cells, orother sample collection device) is provided to a medical practitioner(e.g., a physician) which the medical practitioner uses to obtain asample (e.g., buccal cells, saliva, blood, etc.) from a patient, thesample is then sent to a laboratory (e.g., a CLIA-certified laboratory)or other facility that tests the sample for one or more SNPs disclosedherein (e.g., to determine the genotype of one or more SNPs disclosedherein, such as to determine the patient's predicted response to statintreatment for reducing their risk for CVD, particularly CHD (such as MI)or stroke, and/or their risk for developing CVD), and the results of thetest (e.g., the patient's genotype at one or more SNPs disclosed hereinand/or the patient's predicted statin response or CVD risk based ontheir SNP genotype) are provided back to the medical practitioner(and/or directly to the patient and/or to another party such as ahospital, medical insurance company, genetic counselor, etc.) who maythen provide or otherwise convey the results to the patient. The resultsare typically provided in the form of a report, such as described above.

In certain further exemplary methods of doing business, kits forcollecting a biological sample from a customer (e.g., a buccal swab forcollecting buccal cells, or other sample collection device) are provided(e.g., for sale), such as at an outlet (e.g., a drug store, pharmacy,general merchandise store, or any other desirable outlet), online viathe internet, by mail order, etc., whereby customers can obtain (e.g.,purchase) the kits, collect their own biological samples, and submit(e.g., send/deliver via mail) their samples to a laboratory (e.g., aCLIA-certified laboratory) or other facility which tests the samples forone or more SNPs disclosed herein (e.g., to determine the genotype ofone or more SNPs disclosed herein, such as to determine the customer'spredicted response to statin treatment for reducing their risk for CVD,particularly CHD (e.g., MI) or stroke, and/or their risk for developingCVD) and provides the results of the test (e.g., of the customer'sgenotype at one or more SNPs disclosed herein and/or the customer'sstatin response or CVD risk based on their SNP genotype) back to thecustomer and/or to a third party (e.g., a physician or other medicalpractitioner, hospital, medical insurance company, genetic counselor,etc.). The results are typically provided in the form of a report, suchas described above. If the results of the test are provided to a thirdparty, then this third party may optionally provide another report tothe customer based on the results of the test (e.g., the result of thetest from the laboratory may provide the customer's genotype at one ormore SNPs disclosed herein without statin response or CVD riskinformation, and the third party may provide a report of the customer'sstatin response or CVD risk based on this genotype result).

Certain further embodiments of the invention provide a system fordetermining whether an individual will benefit from statin treatment (orother therapy) in reducing CVD risk (particularly risk for CHD (such asMI) or stroke), or for determining an individual's risk for developingCVD. Certain exemplary systems comprise an integrated “loop” in which anindividual (or their medical practitioner) requests a determination ofsuch individual's predicted statin response (or CVD risk, etc.), thisdetermination is carried out by testing a sample from the individual,and then the results of this determination are provided back to therequestor. For example, in certain systems, a sample (e.g., buccalcells, saliva, blood, etc.) is obtained from an individual for testing(the sample may be obtained by the individual or, for example, by amedical practitioner), the sample is submitted to a laboratory (or otherfacility) for testing (e.g., determining the genotype of one or moreSNPs disclosed herein), and then the results of the testing are sent tothe patient (which optionally can be done by first sending the resultsto an intermediary, such as a medical practioner, who then provides orotherwise conveys the results to the individual and/or acts on theresults), thereby forming an integrated loop system for determining anindividual's predicted statin response (or CVD risk, etc.). The portionsof the system in which the results are transmitted (e.g., between any ofa testing facility, a medical practitioner, and/or the individual) canbe carried out by way of electronic or signal transmission (e.g., bycomputer such as via e-mail or the internet, by providing the results ona website or computer network server which may optionally be a securedatabase, by phone or fax, or by any other wired or wirelesstransmission methods known in the art). Optionally, the system canfurther include a risk reduction component (i.e., a disease managementsystem) as part of the integrated loop (for an example of a diseasemanagement system, see U.S. Pat. No. 6,770,029, “Disease managementsystem and method including correlation assessment”). For example, theresults of the test can be used to reduce the risk of the disease in theindividual who was tested, such as by implementing a preventive therapyregimen (e.g., administration of a statin or other drug for reducing CVDrisk), modifying the individual's diet, increasing exercise, reducingstress, and/or implementing any other physiological or behavioralmodifications in the individual with the goal of reducing disease risk.For reducing CVD risk, this may include any means used in the art forimproving aspects of an individual's health relevant to reducing CVDrisk. Thus, in exemplary embodiments, the system is controlled by theindividual and/or their medical practioner in that the individual and/ortheir medical practioner requests the test, receives the test resultsback, and (optionally) acts on the test results to reduce theindividual's disease risk, such as by implementing a disease managementsystem.

Isolated Nucleic Acid Molecules and SNP Detection Reagents & Kits

Tables 1 and 2 provide a variety of information about each SNP of thepresent invention that is associated with response to statin treatment,particularly for reducing an individual's risk for CVD such as CHD(e.g., MI) or stroke, including the transcript sequences (SEQ IDNOS:1-51), genomic sequences (SEQ ID NOS:177-622), and protein sequences(SEQ ID NOS:52-102) of the encoded gene products (with the SNPsindicated by IUB codes in the nucleic acid sequences). In addition,Tables 1 and 2 include SNP context sequences, which generally include100 nucleotide upstream (5′) plus 100 nucleotides downstream (3′) ofeach SNP position (SEQ ID NOS:103-176 correspond to transcript-based SNPcontext sequences disclosed in Table 1, and SEQ ID NOS:623-3661correspond to genomic-based context sequences disclosed in Table 2), thealternative nucleotides (alleles) at each SNP position, and additionalinformation about the variant where relevant, such as SNP type (coding,missense, splice site, UTR, etc.), human populations in which the SNPwas observed, observed allele frequencies, information about the encodedprotein, etc.

Isolated Nucleic Acid Molecules

Exemplary embodiments of the invention provide isolated nucleic acidmolecules that contain one or more SNPs disclosed herein, particularlySNPs disclosed in Table 1 and/or Table 2. Isolated nucleic acidmolecules containing one or more SNPs disclosed herein (such as in atleast one of Tables 1 and 2) may be interchangeably referred tothroughout the present text as “SNP-containing nucleic acid molecules.”Isolated nucleic acid molecules may optionally encode a full-lengthvariant protein or fragment thereof. The isolated nucleic acid moleculesof the present invention also include probes and primers (which aredescribed in greater detail below in the section entitled “SNP DetectionReagents”), which may be used for assaying the disclosed SNPs, andisolated full-length genes, transcripts, cDNA molecules, and fragmentsthereof, which may be used for such purposes as expressing an encodedprotein.

As used herein, an “isolated nucleic acid molecule” generally is onethat contains a SNP of the present invention or one that hybridizes tosuch molecule such as a nucleic acid with a complementary sequence, andis separated from most other nucleic acids present in the natural sourceof the nucleic acid molecule. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule containing a SNP of the presentinvention, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. A nucleicacid molecule can be fused to other coding or regulatory sequences andstill be considered “isolated.” Nucleic acid molecules present innon-human transgenic animals, which do not naturally occur in theanimal, are also considered “isolated.” For example, recombinant DNAmolecules contained in a vector are considered “isolated.” Furtherexamples of “isolated” DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, and purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the isolated SNP-containing DNAmolecules of the present invention. Isolated nucleic acid moleculesaccording to the present invention further include such moleculesproduced synthetically.

Generally, an isolated SNP-containing nucleic acid molecule comprisesone or more SNP positions disclosed by the present invention withflanking nucleotide sequences on either side of the SNP positions. Aflanking sequence can include nucleotide residues that are naturallyassociated with the SNP site and/or heterologous nucleotide sequences.Preferably, the flanking sequence is up to about 500, 300, 100, 60, 50,30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in-between)on either side of a SNP position, or as long as the full-length gene orentire protein-coding sequence (or any portion thereof such as an exon),especially if the SNP-containing nucleic acid molecule is to be used toproduce a protein or protein fragment.

For full-length genes and entire protein-coding sequences, a SNPflanking sequence can be, for example, up to about 5 KB, 4 KB, 3 KB, 2KB, 1 KB on either side of the SNP. Furthermore, in such instances theisolated nucleic acid molecule comprises exonic sequences (includingprotein-coding and/or non-coding exonic sequences), but may also includeintronic sequences. Thus, any protein coding sequence may be eithercontiguous or separated by introns. The important point is that thenucleic acid is isolated from remote and unimportant flanking sequencesand is of appropriate length such that it can be subjected to thespecific manipulations or uses described herein such as recombinantprotein expression, preparation of probes and primers for assaying theSNP position, and other uses specific to the SNP-containing nucleic acidsequences.

An isolated SNP-containing nucleic acid molecule can comprise, forexample, a full-length gene or transcript, such as a gene isolated fromgenomic DNA (e.g., by cloning or PCR amplification), a cDNA molecule, oran mRNA transcript molecule. Polymorphic transcript sequences arereferred to in Table 1 and provided in the Sequence Listing (SEQ IDNOS:1-51), and polymorphic genomic sequences are referred to in Table 2and provided in the Sequence Listing (SEQ ID NOS:177-622). Furthermore,fragments of such full-length genes and transcripts that contain one ormore SNPs disclosed herein are also encompassed by the presentinvention, and such fragments may be used, for example, to express anypart of a protein, such as a particular functional domain or anantigenic epitope.

Thus, the present invention also encompasses fragments of the nucleicacid sequences as disclosed in Tables 1 and 2 (transcript sequences arereferred to in Table 1 as SEQ ID NOS:1-51, genomic sequences arereferred to in Table 2 as SEQ ID NOS:177-622, transcript-based SNPcontext sequences are referred to in Table 1 as SEQ ID NOS:103-176, andgenomic-based SNP context sequences are referred to in Table 2 as SEQ IDNOS:623-3661) and their complements. The actual sequences referred to inthe tables are provided in the Sequence Listing. A fragment typicallycomprises a contiguous nucleotide sequence at least about 8 or morenucleotides, more preferably at least about 12 or more nucleotides, andeven more preferably at least about 16 or more nucleotides. Furthermore,a fragment could comprise at least about 18, 20, 22, 25, 30, 40, 50, 60,80, 100, 150, 200, 250 or 500 nucleotides in length (or any other numberin between). The length of the fragment will be based on its intendeduse. For example, the fragment can encode epitope-bearing regions of avariant peptide or regions of a variant peptide that differ from thenormal/wild-type protein, or can be useful as a polynucleotide probe orprimer. Such fragments can be isolated using the nucleotide sequencesprovided in Table 1 and/or Table 2 for the synthesis of a polynucleotideprobe. A labeled probe can then be used, for example, to screen a cDNAlibrary, genomic DNA library, or mRNA to isolate nucleic acidcorresponding to the coding region. Further, primers can be used inamplification reactions, such as for purposes of assaying one or moreSNPs sites or for cloning specific regions of a gene.

An isolated nucleic acid molecule of the present invention furtherencompasses a SNP-containing polynucleotide that is the product of anyone of a variety of nucleic acid amplification methods, which are usedto increase the copy numbers of a polynucleotide of interest in anucleic acid sample. Such amplification methods are well known in theart, and they include but are not limited to, polymerase chain reaction(PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Technology:Principles and Applications for DNA Amplification, ed. H. A. Erlich,Freeman Press, NY, NY (1992)), ligase chain reaction (LCR) (Wu andWallace, Genomics 4:560 (1989); Landegren et al., Science 241:1077(1988)), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184 and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923) and the like, and isothermal amplification methods suchas nucleic acid sequence based amplification (NASBA) and self-sustainedsequence replication (Guatelli et al., Proc Natl Acad Sci USA 87:1874(1990)). Based on such methodologies, a person skilled in the art canreadily design primers in any suitable regions 5′ and 3′ to a SNPdisclosed herein. Such primers may be used to amplify DNA of any lengthso long that it contains the SNP of interest in its sequence.

As used herein, an “amplified polynucleotide” of the invention is aSNP-containing nucleic acid molecule whose amount has been increased atleast two fold by any nucleic acid amplification method performed invitro as compared to its starting amount in a test sample. In otherpreferred embodiments, an amplified polynucleotide is the result of atleast ten fold, fifty fold, one hundred fold, one thousand fold, or eventen thousand fold increase as compared to its starting amount in a testsample. In a typical PCR amplification, a polynucleotide of interest isoften amplified at least fifty thousand fold in amount over theunamplified genomic DNA, but the precise amount of amplification neededfor an assay depends on the sensitivity of the subsequent detectionmethod used.

Generally, an amplified polynucleotide is at least about 16 nucleotidesin length. More typically, an amplified polynucleotide is at least about20 nucleotides in length. In a preferred embodiment of the invention, anamplified polynucleotide is at least about 30 nucleotides in length. Ina more preferred embodiment of the invention, an amplifiedpolynucleotide is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In yet another preferred embodiment of the invention, anamplified polynucleotide is at least about 100, 200, 300, 400, or 500nucleotides in length. While the total length of an amplifiedpolynucleotide of the invention can be as long as an exon, an intron orthe entire gene where the SNP of interest resides, an amplified productis typically up to about 1,000 nucleotides in length (although certainamplification methods may generate amplified products greater than 1000nucleotides in length). More preferably, an amplified polynucleotide isnot greater than about 600-700 nucleotides in length. It is understoodthat irrespective of the length of an amplified polynucleotide, a SNP ofinterest may be located anywhere along its sequence.

In a specific embodiment of the invention, the amplified product is atleast about 201 nucleotides in length, comprises one of thetranscript-based context sequences or the genomic-based contextsequences shown in Tables 1 and 2. Such a product may have additionalsequences on its 5′ end or 3′ end or both. In another embodiment, theamplified product is about 101 nucleotides in length, and it contains aSNP disclosed herein. Preferably, the SNP is located at the middle ofthe amplified product (e.g., at position 101 in an amplified productthat is 201 nucleotides in length, or at position 51 in an amplifiedproduct that is 101 nucleotides in length), or within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, or 20 nucleotides from the middle of the amplifiedproduct. However, as indicated above, the SNP of interest may be locatedanywhere along the length of the amplified product.

The present invention provides isolated nucleic acid molecules thatcomprise, consist of, or consist essentially of one or morepolynucleotide sequences that contain one or more SNPs disclosed herein,complements thereof, and SNP-containing fragments thereof.

Accordingly, the present invention provides nucleic acid molecules thatconsist of any of the nucleotide sequences shown in Table 1 and/or Table2 (transcript sequences are referred to in Table 1 as SEQ ID NOS:1-51,genomic sequences are referred to in Table 2 as SEQ ID NOS:177-622,transcript-based SNP context sequences are referred to in Table 1 as SEQID NOS:103-176, and genomic-based SNP context sequences are referred toin Table 2 as SEQ ID NOS:623-3661), or any nucleic acid molecule thatencodes any of the variant proteins referred to in Table 1 (SEQ IDNOS:52-102). The actual sequences referred to in the tables are providedin the Sequence Listing. A nucleic acid molecule consists of anucleotide sequence when the nucleotide sequence is the completenucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of any of the nucleotide sequences referred to inTable 1 and/or Table 2 (transcript sequences are referred to in Table 1as SEQ ID NOS:1-51, genomic sequences are referred to in Table 2 as SEQID NOS:177-622, transcript-based SNP context sequences are referred toin Table 1 as SEQ ID NOS:103-176, and genomic-based SNP contextsequences are referred to in Table 2 as SEQ ID NOS:623-3661), or anynucleic acid molecule that encodes any of the variant proteins referredto in Table 1 (SEQ ID NOS:52-102). The actual sequences referred to inthe tables are provided in the Sequence Listing. A nucleic acid moleculeconsists essentially of a nucleotide sequence when such a nucleotidesequence is present with only a few additional nucleotide residues inthe final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise any of the nucleotide sequences shown in Table 1 and/or Table 2or a SNP-containing fragment thereof (transcript sequences are referredto in Table 1 as SEQ ID NOS:1-51, genomic sequences are referred to inTable 2 as SEQ ID NOS:177-622, transcript-based SNP context sequencesare referred to in Table 1 as SEQ ID NOS:103-176, and genomic-based SNPcontext sequences are referred to in Table 2 as SEQ ID NOS:623-3661), orany nucleic acid molecule that encodes any of the variant proteinsprovided in Table 1 (SEQ ID NOS:52-102). The actual sequences referredto in the tables are provided in the Sequence Listing. A nucleic acidmolecule comprises a nucleotide sequence when the nucleotide sequence isat least part of the final nucleotide sequence of the nucleic acidmolecule. In such a fashion, the nucleic acid molecule can be only thenucleotide sequence or have additional nucleotide residues, such asresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have one to a fewadditional nucleotides or can comprise many more additional nucleotides.A brief description of how various types of these nucleic acid moleculescan be readily made and isolated is provided below, and such techniquesare well known to those of ordinary skill in the art. Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, N.Y. (2000).

The isolated nucleic acid molecules can encode mature proteins plusadditional amino or carboxyl-terminal amino acids or both, or aminoacids interior to the mature peptide (when the mature form has more thanone peptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production. As generally is thecase in situ, the additional amino acids may be processed away from themature protein by cellular enzymes.

Thus, the isolated nucleic acid molecules include, but are not limitedto, nucleic acid molecules having a sequence encoding a peptide alone, asequence encoding a mature peptide and additional coding sequences suchas a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), a sequence encoding a mature peptide with or withoutadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut untranslated sequences that play a role in, for example,transcription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding, and/or stability of mRNA. In addition, thenucleic acid molecules may be fused to heterologous marker sequencesencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA, which may beobtained, for example, by molecular cloning or produced by chemicalsynthetic techniques or by a combination thereof. Sambrook and Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y.(2000). Furthermore, isolated nucleic acid molecules, particularly SNPdetection reagents such as probes and primers, can also be partially orcompletely in the form of one or more types of nucleic acid analogs,such as peptide nucleic acid (PNA). U.S. Pat. Nos. 5,539,082; 5,527,675;5,623,049; and 5,714,331. The nucleic acid, especially DNA, can bedouble-stranded or single-stranded. Single-stranded nucleic acid can bethe coding strand (sense strand) or the complementary non-coding strand(anti-sense strand). DNA, RNA, or PNA segments can be assembled, forexample, from fragments of the human genome (in the case of DNA or RNA)or single nucleotides, short oligonucleotide linkers, or from a seriesof oligonucleotides, to provide a synthetic nucleic acid molecule.Nucleic acid molecules can be readily synthesized using the sequencesprovided herein as a reference; oligonucleotide and PNA oligomersynthesis techniques are well known in the art. See, e.g., Corey,“Peptide nucleic acids: expanding the scope of nucleic acidrecognition,” Trends Biotechnol 15(6):224-9 (June 1997), and Hyrup etal., “Peptide nucleic acids (PNA): synthesis, properties and potentialapplications,” Bioorg Med Chem 4(1):5-23) (January 1996). Furthermore,large-scale automated oligonucleotide/PNA synthesis (including synthesison an array or bead surface or other solid support) can readily beaccomplished using commercially available nucleic acid synthesizers,such as the Applied Biosystems (Foster City, CA) 3900 High-ThroughputDNA Synthesizer or Expedite 8909 Nucleic Acid Synthesis System, and thesequence information provided herein.

The present invention encompasses nucleic acid analogs that containmodified, synthetic, or non-naturally occurring nucleotides orstructural elements or other alternative/modified nucleic acidchemistries known in the art. Such nucleic acid analogs are useful, forexample, as detection reagents (e.g., primers/probes) for detecting oneor more SNPs identified in Table 1 and/or Table 2. Furthermore,kits/systems (such as beads, arrays, etc.) that include these analogsare also encompassed by the present invention. For example, PNAoligomers that are based on the polymorphic sequences of the presentinvention are specifically contemplated. PNA oligomers are analogs ofDNA in which the phosphate backbone is replaced with a peptide-likebackbone. Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal ChemistryLetters 6:793-796 (1996); Kumar et al., Organic Letters 3(9):1269-1272(2001); WO 96/04000. PNA hybridizes to complementary RNA or DNA withhigher affinity and specificity than conventional oligonucleotides andoligonucleotide analogs. The properties of PNA enable novel molecularbiology and biochemistry applications unachievable with traditionaloligonucleotides and peptides.

Additional examples of nucleic acid modifications that improve thebinding properties and/or stability of a nucleic acid include the use ofbase analogs such as inosine, intercalators (U.S. Pat. No. 4,835,263)and the minor groove binders (U.S. Pat. No. 5,801,115). Thus, referencesherein to nucleic acid molecules, SNP-containing nucleic acid molecules,SNP detection reagents (e.g., probes and primers),oligonucleotides/polynucleotides include PNA oligomers and other nucleicacid analogs. Other examples of nucleic acid analogs andalternative/modified nucleic acid chemistries known in the art aredescribed in Current Protocols in Nucleic Acid Chemistry, John Wiley &Sons, N.Y. (2002).

The present invention further provides nucleic acid molecules thatencode fragments of the variant polypeptides disclosed herein as well asnucleic acid molecules that encode obvious variants of such variantpolypeptides. Such nucleic acid molecules may be naturally occurring,such as paralogs (different locus) and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Non-naturally occurring variants may be made by mutagenesistechniques, including those applied to nucleic acid molecules, cells, ororganisms. Accordingly, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions (in addition to theSNPs disclosed in Tables 1 and 2). Variation can occur in either or boththe coding and non-coding regions. The variations can produceconservative and/or non-conservative amino acid substitutions.

Further variants of the nucleic acid molecules disclosed in Tables 1 and2, such as naturally occurring allelic variants (as well as orthologsand paralogs) and synthetic variants produced by mutagenesis techniques,can be identified and/or produced using methods well known in the art.Such further variants can comprise a nucleotide sequence that shares atleast 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity with a nucleic acid sequence disclosed in Table 1and/or Table 2 (or a fragment thereof) and that includes a novel SNPallele disclosed in Table 1 and/or Table 2. Further, variants cancomprise a nucleotide sequence that encodes a polypeptide that shares atleast 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity with a polypeptide sequence disclosed in Table 1(or a fragment thereof) and that includes a novel SNP allele disclosedin Table 1 and/or Table 2. Thus, an aspect of the present invention thatis specifically contemplated are isolated nucleic acid molecules thathave a certain degree of sequence variation compared with the sequencesshown in Tables 1-2, but that contain a novel SNP allele disclosedherein. In other words, as long as an isolated nucleic acid moleculecontains a novel SNP allele disclosed herein, other portions of thenucleic acid molecule that flank the novel SNP allele can vary to somedegree from the specific transcript, genomic, and context sequencesreferred to and shown in Tables 1 and 2, and can encode a polypeptidethat varies to some degree from the specific polypeptide sequencesreferred to in Table 1.

To determine the percent identity of two amino acid sequences or twonucleotide sequences of two molecules that share sequence homology, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, atleast 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of areference sequence is aligned for comparison purposes. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein, amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. Computational Molecular Biology, A. M. Lesk, ed., OxfordUniversity Press, N.Y (1988); Biocomputing: Informatics and GenomeProjects, D. W. Smith, ed., Academic Press, N.Y. (1993); ComputerAnalysis of Sequence Data, Part 1, A. M. Griffin and H. G. Griffin,eds., Humana Press, N.J. (1994); Sequence Analysis in Molecular Biology,G. von Heinje, ed., Academic Press, N.Y. (1987); and Sequence AnalysisPrimer, M. Gribskov and J. Devereux, eds., M. Stockton Press, N.Y.(1991). In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunschalgorithm (J Mol Biol (48):444-453 (1970)) which has been incorporatedinto the GAP program in the GCG software package, using either a Blossom62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

In yet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package using a NWSgapdna. CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. J.Devereux et al., Nucleic Acids Res. 12(1):387 (1984). In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4.

The nucleotide and amino acid sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases; for example, to identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0). Altschul et al., J Mol Biol 215:403-10(1990). BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized. Altschul et al., Nucleic Acids Res25(17):3389-3402 (1997). When utilizing BLAST and gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used. In addition to BLAST, examples of other search andsequence comparison programs used in the art include, but are notlimited to, FASTA (Pearson, Methods Mol Biol 25, 365-389 (1994)) andKERR (Dufresne et al., Nat Biotechnol 20(12):1269-71 (December 2002)).For further information regarding bioinformatics techniques, see CurrentProtocols in Bioinformatics, John Wiley & Sons, Inc., N.Y.

The present invention further provides non-coding fragments of thenucleic acid molecules disclosed in Table 1 and/or Table 2. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, intronic sequences, 5′ untranslatedregions (UTRs), 3′ untranslated regions, gene modulating sequences andgene termination sequences. Such fragments are useful, for example, incontrolling heterologous gene expression and in developing screens toidentify gene-modulating agents.

SNP Detection Reagents

In a specific aspect of the present invention, the SNPs disclosed inTable 1 and/or Table 2, and their associated transcript sequences(referred to in Table 1 as SEQ ID NOS:1-51), genomic sequences (referredto in Table 2 as SEQ ID NOS:177-622), and context sequences(transcript-based context sequences are referred to in Table 1 as SEQ IDNOS:103-176; genomic-based context sequences are provided in Table 2 asSEQ ID NOS:623-3661), can be used for the design of SNP detectionreagents. The actual sequences referred to in the tables are provided inthe Sequence Listing. As used herein, a “SNP detection reagent” is areagent that specifically detects a specific target SNP positiondisclosed herein, and that is preferably specific for a particularnucleotide (allele) of the target SNP position (i.e., the detectionreagent preferably can differentiate between different alternativenucleotides at a target SNP position, thereby allowing the identity ofthe nucleotide present at the target SNP position to be determined).Typically, such detection reagent hybridizes to a target SNP-containingnucleic acid molecule by complementary base-pairing in a sequencespecific manner, and discriminates the target variant sequence fromother nucleic acid sequences such as an art-known form in a test sample.An example of a detection reagent is a probe that hybridizes to a targetnucleic acid containing one or more of the SNPs referred to in Table 1and/or Table 2. In a preferred embodiment, such a probe candifferentiate between nucleic acids having a particular nucleotide(allele) at a target SNP position from other nucleic acids that have adifferent nucleotide at the same target SNP position. In addition, adetection reagent may hybridize to a specific region 5′ and/or 3′ to aSNP position, particularly a region corresponding to the contextsequences referred to in Table 1 and/or Table 2 (transcript-basedcontext sequences are referred to in Table 1 as SEQ ID NOS:103-176;genomic-based context sequences are referred to in Table 2 as SEQ IDNOS:623-3661). Another example of a detection reagent is a primer thatacts as an initiation point of nucleotide extension along acomplementary strand of a target polynucleotide. The SNP sequenceinformation provided herein is also useful for designing primers, e.g.allele-specific primers, to amplify (e.g., using PCR) any SNP of thepresent invention.

In one preferred embodiment of the invention, a SNP detection reagent isan isolated or synthetic DNA or RNA polynucleotide probe or primer orPNA oligomer, or a combination of DNA, RNA and/or PNA, that hybridizesto a segment of a target nucleic acid molecule containing a SNPidentified in Table 1 and/or Table 2. A detection reagent in the form ofa polynucleotide may optionally contain modified base analogs,intercalators or minor groove binders. Multiple detection reagents suchas probes may be, for example, affixed to a solid support (e.g., arraysor beads) or supplied in solution (e.g. probe/primer sets for enzymaticreactions such as PCR, RT-PCR, TaqMan assays, or primer-extensionreactions) to form a SNP detection kit.

A probe or primer typically is a substantially purified oligonucleotideor PNA oligomer. Such oligonucleotide typically comprises a region ofcomplementary nucleotide sequence that hybridizes under stringentconditions to at least about 8, 10, 12, 16, 18, 20, 22, 25, 30, 40, 50,55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) ormore consecutive nucleotides in a target nucleic acid molecule.Depending on the particular assay, the consecutive nucleotides caneither include the target SNP position, or be a specific region in closeenough proximity 5′ and/or 3′ to the SNP position to carry out thedesired assay.

Other preferred primer and probe sequences can readily be determinedusing the transcript sequences (SEQ ID NOS:1-51), genomic sequences (SEQID NOS:177-622), and SNP context sequences (transcript-based contextsequences are referred to in Table 1 as SEQ ID NOS:103-176;genomic-based context sequences are referred to in Table 2 as SEQ IDNOS:623-3661) disclosed in the Sequence Listing and in Tables 1 and 2.The actual sequences referred to in the tables are provided in theSequence Listing. It will be apparent to one of skill in the art thatsuch primers and probes are directly useful as reagents for genotypingthe SNPs of the present invention, and can be incorporated into anykit/system format.

In order to produce a probe or primer specific for a targetSNP-containing sequence, the gene/transcript and/or context sequencesurrounding the SNP of interest is typically examined using a computeralgorithm that starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene/SNP context sequence, have a GCcontent within a range suitable for hybridization, lack predictedsecondary structure that may interfere with hybridization, and/orpossess other desired characteristics or that lack other undesiredcharacteristics.

A primer or probe of the present invention is typically at least about 8nucleotides in length. In one embodiment of the invention, a primer or aprobe is at least about 10 nucleotides in length. In a preferredembodiment, a primer or a probe is at least about 12 nucleotides inlength. In a more preferred embodiment, a primer or probe is at leastabout 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.While the maximal length of a probe can be as long as the targetsequence to be detected, depending on the type of assay in which it isemployed, it is typically less than about 50, 60, 65, or 70 nucleotidesin length. In the case of a primer, it is typically less than about 30nucleotides in length. In a specific preferred embodiment of theinvention, a primer or a probe is within the length of about 18 andabout 28 nucleotides. However, in other embodiments, such as nucleicacid arrays and other embodiments in which probes are affixed to asubstrate, the probes can be longer, such as on the order of 30-70, 75,80, 90, 100, or more nucleotides in length (see the section belowentitled “SNP Detection Kits and Systems”).

For analyzing SNPs, it may be appropriate to use oligonucleotidesspecific for alternative SNP alleles. Such oligonucleotides that detectsingle nucleotide variations in target sequences may be referred to bysuch terms as “allele-specific oligonucleotides,” “allele-specificprobes,” or “allele-specific primers.” The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection: A Practical Approach, Cotton et al., eds.,Oxford University Press (1998); Saiki et al., Nature 324:163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548.

While the design of each allele-specific primer or probe depends onvariables such as the precise composition of the nucleotide sequencesflanking a SNP position in a target nucleic acid molecule, and thelength of the primer or probe, another factor in the use of primers andprobes is the stringency of the condition under which the hybridizationbetween the probe or primer and the target sequence is performed. Higherstringency conditions utilize buffers with lower ionic strength and/or ahigher reaction temperature, and tend to require a more perfect matchbetween probe/primer and a target sequence in order to form a stableduplex. If the stringency is too high, however, hybridization may notoccur at all. In contrast, lower stringency conditions utilize bufferswith higher ionic strength and/or a lower reaction temperature, andpermit the formation of stable duplexes with more mismatched basesbetween a probe/primer and a target sequence. By way of example and notlimitation, exemplary conditions for high stringency hybridizationconditions using an allele-specific probe are as follows:prehybridization with a solution containing 5× standard saline phosphateEDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubating probe withtarget nucleic acid molecules in the same solution at the sametemperature, followed by washing with a solution containing 2×SSPE, and0.1% SDS at 55° C. or room temperature.

Moderate stringency hybridization conditions may be used forallele-specific primer extension reactions with a solution containing,e.g., about 50 mM KCl at about 46° C. Alternatively, the reaction may becarried out at an elevated temperature such as 60° C. In anotherembodiment, a moderately stringent hybridization condition suitable foroligonucleotide ligation assay (OLA) reactions wherein two probes areligated if they are completely complementary to the target sequence mayutilize a solution of about 100 mM KCl at a temperature of 46° C.

In a hybridization-based assay, allele-specific probes can be designedthat hybridize to a segment of target DNA from one individual but do nothybridize to the corresponding segment from another individual due tothe presence of different polymorphic forms (e.g., alternative SNPalleles/nucleotides) in the respective DNA segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant detectable difference in hybridizationintensity between alleles, and preferably an essentially binaryresponse, whereby a probe hybridizes to only one of the alleles orsignificantly more strongly to one allele. While a probe may be designedto hybridize to a target sequence that contains a SNP site such that theSNP site aligns anywhere along the sequence of the probe, the probe ispreferably designed to hybridize to a segment of the target sequencesuch that the SNP site aligns with a central position of the probe(e.g., a position within the probe that is at least three nucleotidesfrom either end of the probe). This design of probe generally achievesgood discrimination in hybridization between different allelic forms.

In another embodiment, a probe or primer may be designed to hybridize toa segment of target DNA such that the SNP aligns with either the 5′ mostend or the 3′ most end of the probe or primer. In a specific preferredembodiment that is particularly suitable for use in a oligonucleotideligation assay (U.S. Pat. No. 4,988,617), the 3′ most nucleotide of theprobe aligns with the SNP position in the target sequence.

Oligonucleotide probes and primers may be prepared by methods well knownin the art. Chemical synthetic methods include, but are not limited to,the phosphotriester method described by Narang et al., Methods inEnzymology 68:90 (1979); the phosphodiester method described by Brown etal., Methods in Enzymology 68:109 (1979); the diethylphosphoamidatemethod described by Beaucage et al., Tetrahedron Letters 22:1859 (1981);and the solid support method described in U.S. Pat. No. 4,458,066.

Allele-specific probes are often used in pairs (or, less commonly, insets of 3 or 4, such as if a SNP position is known to have 3 or 4alleles, respectively, or to assay both strands of a nucleic acidmolecule for a target SNP allele), and such pairs may be identicalexcept for a one nucleotide mismatch that represents the allelicvariants at the SNP position. Commonly, one member of a pair perfectlymatches a reference form of a target sequence that has a more common SNPallele (i.e., the allele that is more frequent in the target population)and the other member of the pair perfectly matches a form of the targetsequence that has a less common SNP allele (i.e., the allele that israrer in the target population). In the case of an array, multiple pairsof probes can be immobilized on the same support for simultaneousanalysis of multiple different polymorphisms.

In one type of PCR-based assay, an allele-specific primer hybridizes toa region on a target nucleic acid molecule that overlaps a SNP positionand only primes amplification of an allelic form to which the primerexhibits perfect complementarity. Gibbs, Nucleic Acid Res 17:2427-2448(1989). Typically, the primer's 3′-most nucleotide is aligned with andcomplementary to the SNP position of the target nucleic acid molecule.This primer is used in conjunction with a second primer that hybridizesat a distal site. Amplification proceeds from the two primers, producinga detectable product that indicates which allelic form is present in thetest sample. A control is usually performed with a second pair ofprimers, one of which shows a single base mismatch at the polymorphicsite and the other of which exhibits perfect complementarity to a distalsite. The single-base mismatch prevents amplification or substantiallyreduces amplification efficiency, so that either no detectable productis formed or it is formed in lower amounts or at a slower pace. Themethod generally works most effectively when the mismatch is at the3′-most position of the oligonucleotide (i.e., the 3′-most position ofthe oligonucleotide aligns with the target SNP position) because thisposition is most destabilizing to elongation from the primer (see, e.g.,WO 93/22456). This PCR-based assay can be utilized as part of the TaqManassay, described below.

In a specific embodiment of the invention, a primer of the inventioncontains a sequence substantially complementary to a segment of a targetSNP-containing nucleic acid molecule except that the primer has amismatched nucleotide in one of the three nucleotide positions at the3′-most end of the primer, such that the mismatched nucleotide does notbase pair with a particular allele at the SNP site. In a preferredembodiment, the mismatched nucleotide in the primer is the second fromthe last nucleotide at the 3′-most position of the primer. In a morepreferred embodiment, the mismatched nucleotide in the primer is thelast nucleotide at the 3′-most position of the primer.

In another embodiment of the invention, a SNP detection reagent of theinvention is labeled with a fluorogenic reporter dye that emits adetectable signal. While the preferred reporter dye is a fluorescentdye, any reporter dye that can be attached to a detection reagent suchas an oligonucleotide probe or primer is suitable for use in theinvention. Such dyes include, but are not limited to, Acridine, AMCA,BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin,Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine,Rhodol Green, Tamra, Rox, and Texas Red.

In yet another embodiment of the invention, the detection reagent may befurther labeled with a quencher dye such as Tamra, especially when thereagent is used as a self-quenching probe such as a TaqMan (U.S. Pat.Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos.5,118,801 and 5,312,728), or other stemless or linear beacon probe(Livak et al., PCR Method Appl 4:357-362 (1995); Tyagi et al., NatureBiotechnology 14:303-308 (1996); Nazarenko et al., Nucl Acids Res25:2516-2521 (1997); U.S. Pat. Nos. 5,866,336 and 6,117,635.

The detection reagents of the invention may also contain other labels,including but not limited to, biotin for streptavidin binding, haptenfor antibody binding, and oligonucleotide for binding to anothercomplementary oligonucleotide such as pairs of zipcodes.

The present invention also contemplates reagents that do not contain (orthat are complementary to) a SNP nucleotide identified herein but thatare used to assay one or more SNPs disclosed herein. For example,primers that flank, but do not hybridize directly to a target SNPposition provided herein are useful in primer extension reactions inwhich the primers hybridize to a region adjacent to the target SNPposition (i.e., within one or more nucleotides from the target SNPsite). During the primer extension reaction, a primer is typically notable to extend past a target SNP site if a particular nucleotide(allele) is present at that target SNP site, and the primer extensionproduct can be detected in order to determine which SNP allele ispresent at the target SNP site. For example, particular ddNTPs aretypically used in the primer extension reaction to terminate primerextension once a ddNTP is incorporated into the extension product (aprimer extension product which includes a ddNTP at the 3′-most end ofthe primer extension product, and in which the ddNTP is a nucleotide ofa SNP disclosed herein, is a composition that is specificallycontemplated by the present invention). Thus, reagents that bind to anucleic acid molecule in a region adjacent to a SNP site and that areused for assaying the SNP site, even though the bound sequences do notnecessarily include the SNP site itself, are also contemplated by thepresent invention.

SNP Detection Kits and Systems

A person skilled in the art will recognize that, based on the SNP andassociated sequence information disclosed herein, detection reagents canbe developed and used to assay any SNP of the present inventionindividually or in combination, and such detection reagents can bereadily incorporated into one of the established kit or system formatswhich are well known in the art. The terms “kits” and “systems,” as usedherein in the context of SNP detection reagents, are intended to referto such things as combinations of multiple SNP detection reagents, orone or more SNP detection reagents in combination with one or more othertypes of elements or components (e.g., other types of biochemicalreagents, containers, packages such as packaging intended for commercialsale, substrates to which SNP detection reagents are attached,electronic hardware components, etc.). Accordingly, the presentinvention further provides SNP detection kits and systems, including butnot limited to, packaged probe and primer sets (e.g. TaqMan probe/primersets), arrays/microarrays of nucleic acid molecules, and beads thatcontain one or more probes, primers, or other detection reagents fordetecting one or more SNPs of the present invention. The kits/systemscan optionally include various electronic hardware components; forexample, arrays (“DNA chips”) and microfluidic systems (“lab-on-a-chip”systems) provided by various manufacturers typically comprise hardwarecomponents. Other kits/systems (e.g., probe/primer sets) may not includeelectronic hardware components, but may be comprised of, for example,one or more SNP detection reagents (along with, optionally, otherbiochemical reagents) packaged in one or more containers.

In some embodiments, a SNP detection kit typically contains one or moredetection reagents and other components (e.g. a buffer, enzymes such asDNA polymerases or ligases, chain extension nucleotides such asdeoxynucleotide triphosphates, and in the case of Sanger-type DNAsequencing reactions, chain terminating nucleotides, positive controlsequences, negative control sequences, and the like) necessary to carryout an assay or reaction, such as amplification and/or detection of aSNP-containing nucleic acid molecule. A kit may further contain meansfor determining the amount of a target nucleic acid, and means forcomparing the amount with a standard, and can comprise instructions forusing the kit to detect the SNP-containing nucleic acid molecule ofinterest. In one embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out one or more assays todetect one or more SNPs disclosed herein. In a preferred embodiment ofthe present invention, SNP detection kits/systems are in the form ofnucleic acid arrays, or compartmentalized kits, includingmicrofluidic/lab-on-a-chip systems.

Exemplary kits of the invention can comprise a container containing aSNP detection reagent which detects a SNP disclosed herein, saidcontainer can optionally be enclosed in a package (e.g., a box forcommercial sale), and said package can further include other containerscontaining any or all of the following: enzyme (e.g., polymerase orligase, any of which can be thermostable), dNTPs and/or ddNTPs (whichcan optionally be detectably labeled, such as with a fluorescent labelor mass tag, and such label can optionally differ between any of thedATPs, dCTPs, dGTPs, dTTPs, ddATPs, ddCTPs, ddGTPs, and/or ddTTPs, sothat each of these dNTPs and/or ddNTPs can be distinguished from eachother by detection of the label, and any of these dNTPs and/or ddNTPscan optionally be stored in the same container or each in separatecontainers), buffer, controls (e.g., positive control nucleic acid, or anegative control), reagent(s) for extracting nucleic acid from a testsample, and instructions for using the kit (such as instructions forcorrelating the presence or absence of a particular allele or genotypewith an increased or decreased risk for disease such as CVD, or anincreased or decreased likelihood of responding to a drug such as astatin). The SNP detection reagent can comprise, for example, at leastone primer and/or probe, any of which can optionally be allele-specific,and any of which can optionally be detectably labeled (e.g., with afluorescent label).

SNP detection kits/systems may contain, for example, one or more probes,or pairs of probes, that hybridize to a nucleic acid molecule at or neareach target SNP position. Multiple pairs of allele-specific probes maybe included in the kit/system to simultaneously assay large numbers ofSNPs, at least one of which is a SNP of the present invention. In somekits/systems, the allele-specific probes are immobilized to a substratesuch as an array or bead. For example, the same substrate can compriseallele-specific probes for detecting at least 1; 10; 100; 1000; 10,000;100,000 (or any other number in-between) or substantially all of theSNPs shown in Table 1 and/or Table 2.

The terms “arrays,” “microarrays,” and “DNA chips” are used hereininterchangeably to refer to an array of distinct polynucleotides affixedto a substrate, such as glass, plastic, paper, nylon or other type ofmembrane, filter, chip, or any other suitable solid support. Thepolynucleotides can be synthesized directly on the substrate, orsynthesized separate from the substrate and then affixed to thesubstrate. In one embodiment, the microarray is prepared and usedaccording to the methods described in Chee et al., U.S. Pat. No.5,837,832 and PCT application WO95/11995; D. J. Lockhart et al., NatBiotech 14:1675-1680 (1996); and M. Schena et al., Proc Natl Acad Sci93:10614-10619 (1996), all of which are incorporated herein in theirentirety by reference. In other embodiments, such arrays are produced bythe methods described by Brown et al., U.S. Pat. No. 5,807,522.

Nucleic acid arrays are reviewed in the following references: Zammatteoet al., “New chips for molecular biology and diagnostics,” BiotechnolAnnu Rev 8:85-101 (2002); Sosnowski et al., “Active microelectronicarray system for DNA hybridization, genotyping and pharmacogenomicapplications,” Psychiatr Genet 12(4):181-92 (December 2002); Heller,“DNA microarray technology: devices, systems, and applications,” AnnuRev Biomed Eng 4:129-53 (2002); Epub Mar. 22, 2002; Kolchinsky et al.,“Analysis of SNPs and other genomic variations using gel-based chips,”Hum Mutat 19(4):343-60 (April 2002); and McGall et al., “High-densitygenechip oligonucleotide probe arrays,” Adv Biochem Eng Biotechnol77:21-42 (2002).

Any number of probes, such as allele-specific probes, may be implementedin an array, and each probe or pair of probes can hybridize to adifferent SNP position. In the case of polynucleotide probes, they canbe synthesized at designated areas (or synthesized separately and thenaffixed to designated areas) on a substrate using a light-directedchemical process. Each DNA chip can contain, for example, thousands tomillions of individual synthetic polynucleotide probes arranged in agrid-like pattern and miniaturized (e.g., to the size of a dime).Preferably, probes are attached to a solid support in an ordered,addressable array.

A microarray can be composed of a large number of unique,single-stranded polynucleotides, usually either synthetic antisensepolynucleotides or fragments of cDNAs, fixed to a solid support. Typicalpolynucleotides are preferably about 6-60 nucleotides in length, morepreferably about 15-30 nucleotides in length, and most preferably about18-25 nucleotides in length. For certain types of microarrays or otherdetection kits/systems, it may be preferable to use oligonucleotidesthat are only about 7-20 nucleotides in length. In other types ofarrays, such as arrays used in conjunction with chemiluminescentdetection technology, preferred probe lengths can be, for example, about15-80 nucleotides in length, preferably about 50-70 nucleotides inlength, more preferably about 55-65 nucleotides in length, and mostpreferably about 60 nucleotides in length. The microarray or detectionkit can contain polynucleotides that cover the known 5′ or 3′ sequenceof a gene/transcript or target SNP site, sequential polynucleotides thatcover the full-length sequence of a gene/transcript; or uniquepolynucleotides selected from particular areas along the length of atarget gene/transcript sequence, particularly areas corresponding to oneor more SNPs disclosed in Table 1 and/or Table 2. Polynucleotides usedin the microarray or detection kit can be specific to a SNP or SNPs ofinterest (e.g., specific to a particular SNP allele at a target SNPsite, or specific to particular SNP alleles at multiple different SNPsites), or specific to a polymorphic gene/transcript orgenes/transcripts of interest.

Hybridization assays based on polynucleotide arrays rely on thedifferences in hybridization stability of the probes to perfectlymatched and mismatched target sequence variants. For SNP genotyping, itis generally preferable that stringency conditions used in hybridizationassays are high enough such that nucleic acid molecules that differ fromone another at as little as a single SNP position can be differentiated(e.g., typical SNP hybridization assays are designed so thathybridization will occur only if one particular nucleotide is present ata SNP position, but will not occur if an alternative nucleotide ispresent at that SNP position). Such high stringency conditions may bepreferable when using, for example, nucleic acid arrays ofallele-specific probes for SNP detection. Such high stringencyconditions are described in the preceding section, and are well known tothose skilled in the art and can be found in, for example, CurrentProtocols in Molecular Biology 6.3.1-6.3.6, John Wiley & Sons, N.Y.(1989).

In other embodiments, the arrays are used in conjunction withchemiluminescent detection technology. The following patents and patentapplications, which are all hereby incorporated by reference, provideadditional information pertaining to chemiluminescent detection. U.S.patent applications that describe chemiluminescent approaches formicroarray detection: Ser. Nos. 10/620,332 and 10/620,333. U.S. patentsthat describe methods and compositions of dioxetane for performingchemiluminescent detection: U.S. Pat. Nos. 6,124,478; 6,107,024;5,994,073; 5,981,768; 5,871,938; 5,843,681; 5,800,999 and 5,773,628. Andthe U.S. published application that discloses methods and compositionsfor microarray controls: US2002/0110828.

In one embodiment of the invention, a nucleic acid array can comprise anarray of probes of about 15-25 nucleotides in length. In furtherembodiments, a nucleic acid array can comprise any number of probes, inwhich at least one probe is capable of detecting one or more SNPsdisclosed in Table 1 and/or Table 2, and/or at least one probe comprisesa fragment of one of the sequences selected from the group consisting ofthose disclosed in Table 1, Table 2, the Sequence Listing, and sequencescomplementary thereto, said fragment comprising at least about 8consecutive nucleotides, preferably 10, 12, 15, 16, 18, 20, morepreferably 22, 25, 30, 40, 47, 50, 55, 60, 65, 70, 80, 90, 100, or moreconsecutive nucleotides (or any other number in-between) and containing(or being complementary to) a novel SNP allele disclosed in Table 1and/or Table 2. In some embodiments, the nucleotide complementary to theSNP site is within 5, 4, 3, 2, or 1 nucleotide from the center of theprobe, more preferably at the center of said probe.

A polynucleotide probe can be synthesized on the surface of thesubstrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more polynucleotides, or any other numberwhich lends itself to the efficient use of commercially availableinstrumentation.

Using such arrays or other kits/systems, the present invention providesmethods of identifying the SNPs disclosed herein in a test sample. Suchmethods typically involve incubating a test sample of nucleic acids withan array comprising one or more probes corresponding to at least one SNPposition of the present invention, and assaying for binding of a nucleicacid from the test sample with one or more of the probes. Conditions forincubating a SNP detection reagent (or a kit/system that employs one ormore such SNP detection reagents) with a test sample vary. Incubationconditions depend on such factors as the format employed in the assay,the detection methods employed, and the type and nature of the detectionreagents used in the assay. One skilled in the art will recognize thatany one of the commonly available hybridization, amplification and arrayassay formats can readily be adapted to detect the SNPs disclosedherein.

A SNP detection kit/system of the present invention may includecomponents that are used to prepare nucleic acids from a test sample forthe subsequent amplification and/or detection of a SNP-containingnucleic acid molecule. Such sample preparation components can be used toproduce nucleic acid extracts (including DNA and/or RNA), proteins ormembrane extracts from any bodily fluids (such as blood, serum, plasma,urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin,hair, cells (especially nucleated cells) such as buccal cells (e.g., asobtained by buccal swabs), biopsies, or tissue specimens. The testsamples used in the above-described methods will vary based on suchfactors as the assay format, nature of the detection method, and thespecific tissues, cells or extracts used as the test sample to beassayed. Methods of preparing nucleic acids, proteins, and cell extractsare well known in the art and can be readily adapted to obtain a samplethat is compatible with the system utilized. Automated samplepreparation systems for extracting nucleic acids from a test sample arecommercially available, and examples are Qiagen's BioRobot 9600, AppliedBiosystems' PRISM™ 6700 sample preparation system, and Roche MolecularSystems' COBAS AmpliPrep System.

Another form of kit contemplated by the present invention is acompartmentalized kit. A compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers include,for example, small glass containers, plastic containers, strips ofplastic, glass or paper, or arraying material such as silica. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the test samples andreagents are not cross-contaminated, or from one container to anothervessel not included in the kit, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother or to another vessel. Such containers may include, for example,one or more containers which will accept the test sample, one or morecontainers which contain at least one probe or other SNP detectionreagent for detecting one or more SNPs of the present invention, one ormore containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and one or more containers which containthe reagents used to reveal the presence of the bound probe or other SNPdetection reagents. The kit can optionally further comprise compartmentsand/or reagents for, for example, nucleic acid amplification or otherenzymatic reactions such as primer extension reactions, hybridization,ligation, electrophoresis (preferably capillary electrophoresis), massspectrometry, and/or laser-induced fluorescent detection. The kit mayalso include instructions for using the kit. Exemplary compartmentalizedkits include microfluidic devices known in the art. See, e.g., Weigl etal., “Lab-on-a-chip for drug development,” Adv Drug Deliv Rev55(3):349-77 (February 2003). In such microfluidic devices, thecontainers may be referred to as, for example, microfluidic“compartments,” “chambers,” or “channels.”

Microfluidic devices, which may also be referred to as “lab-on-a-chip”systems, biomedical micro-electro-mechanical systems (bioMEMs), ormulticomponent integrated systems, are exemplary kits/systems of thepresent invention for analyzing SNPs. Such systems miniaturize andcompartmentalize processes such as probe/target hybridization, nucleicacid amplification, and capillary electrophoresis reactions in a singlefunctional device. Such microfluidic devices typically utilize detectionreagents in at least one aspect of the system, and such detectionreagents may be used to detect one or more SNPs of the presentinvention. One example of a microfluidic system is disclosed in U.S.Pat. No. 5,589,136, which describes the integration of PCR amplificationand capillary electrophoresis in chips. Exemplary microfluidic systemscomprise a pattern of microchannels designed onto a glass, silicon,quartz, or plastic wafer included on a microchip. The movements of thesamples may be controlled by electric, electroosmotic or hydrostaticforces applied across different areas of the microchip to createfunctional microscopic valves and pumps with no moving parts. Varyingthe voltage can be used as a means to control the liquid flow atintersections between the micro-machined channels and to change theliquid flow rate for pumping across different sections of the microchip.See, for example, U.S. Pat. No. 6,153,073, Dubrow et al., and U.S. Pat.No. 6,156,181, Parce et al.

For genotyping SNPs, an exemplary microfluidic system may integrate, forexample, nucleic acid amplification, primer extension, capillaryelectrophoresis, and a detection method such as laser inducedfluorescence detection. In a first step of an exemplary process forusing such an exemplary system, nucleic acid samples are amplified,preferably by PCR. Then, the amplification products are subjected toautomated primer extension reactions using ddNTPs (specific fluorescencefor each ddNTP) and the appropriate oligonucleotide primers to carry outprimer extension reactions which hybridize just upstream of the targetedSNP. Once the extension at the 3′ end is completed, the primers areseparated from the unincorporated fluorescent ddNTPs by capillaryelectrophoresis. The separation medium used in capillary electrophoresiscan be, for example, polyacrylamide, polyethyleneglycol or dextran. Theincorporated ddNTPs in the single nucleotide primer extension productsare identified by laser-induced fluorescence detection. Such anexemplary microchip can be used to process, for example, at least 96 to384 samples, or more, in parallel.

Uses of Nucleic Acid Molecules

The nucleic acid molecules of the present invention have a variety ofuses, particularly for predicting whether an individual will benefitfrom statin treatment by reducing their risk for CVD (particularly CHD,such as MI) in response to the statin treatment, as well as for thediagnosis, prognosis, treatment, and prevention of CVD (particularlyCHD, such as MI). For example, the nucleic acid molecules of theinvention are useful for determining the likelihood of an individual whocurrently or previously has or has had CVD (such as an individual whohas previously had an MI) or who is at increased risk for developing CVD(such as an individual who has not yet had an MI but is at increasedrisk for having an MI in the future) of responding to treatment (orprevention) of CVD with statins (such as by reducing their risk ofdeveloping primary or recurrent CVD, such as MI, in the future),predicting the likelihood that the individual will experience toxicityor other undesirable side effects from the statin treatment, predictingan individual's risk for developing CVD (particularly the risk for CHDsuch as MI), etc. For example, the nucleic acid molecules are useful ashybridization probes, such as for genotyping SNPs in messenger RNA,transcript, cDNA, genomic DNA, amplified DNA or other nucleic acidmolecules, and for isolating full-length cDNA and genomic clonesencoding the variant peptides disclosed in Table 1 as well as theirorthologs.

A probe can hybridize to any nucleotide sequence along the entire lengthof a nucleic acid molecule referred to in Table 1 and/or Table 2.Preferably, a probe of the present invention hybridizes to a region of atarget sequence that encompasses a SNP position indicated in Table 1and/or Table 2. More preferably, a probe hybridizes to a SNP-containingtarget sequence in a sequence-specific manner such that it distinguishesthe target sequence from other nucleotide sequences which vary from thetarget sequence only by which nucleotide is present at the SNP site.Such a probe is particularly useful for detecting the presence of aSNP-containing nucleic acid in a test sample, or for determining whichnucleotide (allele) is present at a particular SNP site (i.e.,genotyping the SNP site).

A nucleic acid hybridization probe may be used for determining thepresence, level, form, and/or distribution of nucleic acid expression.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes specific for the SNPs described herein can be usedto assess the presence, expression and/or gene copy number in a givencell, tissue, or organism. These uses are relevant for diagnosis ofdisorders involving an increase or decrease in gene expression relativeto normal levels. In vitro techniques for detection of mRNA include, forexample, Northern blot hybridizations and in situ hybridizations. Invitro techniques for detecting DNA include Southern blot hybridizationsand in situ hybridizations. Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, N.Y. (2000).

Probes can be used as part of a diagnostic test kit for identifyingcells or tissues in which a variant protein is expressed, such as bymeasuring the level of a variant protein-encoding nucleic acid (e.g.,mRNA) in a sample of cells from a subject or determining if apolynucleotide contains a SNP of interest.

Thus, the nucleic acid molecules of the invention can be used ashybridization probes to detect the SNPs disclosed herein, therebydetermining the likelihood that an individual will respond positively tostatin treatment for reducing the risk of CVD (particularly CHD such asMI), or whether an individual with the polymorphism(s) is at risk fordeveloping CVD (or has already developed early stage CVD). Detection ofa SNP associated with a disease phenotype provides a diagnostic tool foran active disease and/or genetic predisposition to the disease.

Furthermore, the nucleic acid molecules of the invention are thereforeuseful for detecting a gene (gene information is disclosed in Table 2,for example) which contains a SNP disclosed herein and/or products ofsuch genes, such as expressed mRNA transcript molecules (transcriptinformation is disclosed in Table 1, for example), and are thus usefulfor detecting gene expression. The nucleic acid molecules can optionallybe implemented in, for example, an array or kit format for use indetecting gene expression.

The nucleic acid molecules of the invention are also useful as primersto amplify any given region of a nucleic acid molecule, particularly aregion containing a SNP identified in Table 1 and/or Table 2.

The nucleic acid molecules of the invention are also useful forconstructing recombinant vectors (described in greater detail below).Such vectors include expression vectors that express a portion of, orall of, any of the variant peptide sequences referred to in Table 1.Vectors also include insertion vectors, used to integrate into anothernucleic acid molecule sequence, such as into the cellular genome, toalter in situ expression of a gene and/or gene product. For example, anendogenous coding sequence can be replaced via homologous recombinationwith all or part of the coding region containing one or morespecifically introduced SNPs.

The nucleic acid molecules of the invention are also useful forexpressing antigenic portions of the variant proteins, particularlyantigenic portions that contain a variant amino acid sequence (e.g., anamino acid substitution) caused by a SNP disclosed in Table 1 and/orTable 2.

The nucleic acid molecules of the invention are also useful forconstructing vectors containing a gene regulatory region of the nucleicacid molecules of the present invention.

The nucleic acid molecules of the invention are also useful fordesigning ribozymes corresponding to all, or a part, of an mRNA moleculeexpressed from a SNP-containing nucleic acid molecule described herein.

The nucleic acid molecules of the invention are also useful forconstructing host cells expressing a part, or all, of the nucleic acidmolecules and variant peptides.

The nucleic acid molecules of the invention are also useful forconstructing transgenic animals expressing all, or a part, of thenucleic acid molecules and variant peptides. The production ofrecombinant cells and transgenic animals having nucleic acid moleculeswhich contain the SNPs disclosed in Table 1 and/or Table 2 allows, forexample, effective clinical design of treatment compounds and dosageregimens.

The nucleic acid molecules of the invention are also useful in assaysfor drug screening to identify compounds that, for example, modulatenucleic acid expression.

The nucleic acid molecules of the invention are also useful in genetherapy in patients whose cells have aberrant gene expression. Thus,recombinant cells, which include a patient's cells that have beenengineered ex vivo and returned to the patient, can be introduced intoan individual where the recombinant cells produce the desired protein totreat the individual.

SNP Genotyping Methods

The process of determining which nucleotide(s) is/are present at each ofone or more SNP positions (such as a SNP position disclosed in Table 1and/or Table 2), for either or both alleles, may be referred to by suchphrases as SNP genotyping, determining the “identity” of a SNP,determining the “content” of a SNP, or determining whichnucleotide(s)/allele(s) is/are present at a SNP position. Thus, theseterms can refer to detecting a single allele (nucleotide) at a SNPposition or can encompass detecting both alleles (nucleotides) at a SNPposition (such as to determine the homozygous or heterozygous state of aSNP position). Furthermore, these terms may also refer to detecting anamino acid residue encoded by a SNP (such as alternative amino acidresidues that are encoded by different codons created by alternativenucleotides at a missense SNP position, for example).

The present invention provides methods of SNP genotyping, such as foruse in implementing a preventive or treatment regimen for an individualbased on that individual having an increased susceptibility fordeveloping CVD (e.g., increased risk for CHD, such as MI) and/or anincreased likelihood of benefiting from statin treatment for reducingthe risk of CVD, in evaluating an individual's likelihood of respondingto statin treatment (particularly for treating or preventing CVD), inselecting a treatment or preventive regimen (e.g., in deciding whetheror not to administer statin treatment to an individual having CVD, orwho is at increased risk for developing CVD, such as MI, in the future),or in formulating or selecting a particular statin-based treatment orpreventive regimen such as dosage and/or frequency of administration ofstatin treatment or choosing which form/type of statin to beadministered, such as a particular pharmaceutical composition orcompound, etc.), determining the likelihood of experiencing toxicity orother undesirable side effects from statin treatment, or selectingindividuals for a clinical trial of a statin (e.g., selectingindividuals to participate in the trial who are most likely to respondpositively from the statin treatment and/or excluding individuals fromthe trial who are unlikely to respond positively from the statintreatment based on their SNP genotype(s), or selecting individuals whoare unlikely to respond positively to statins based on their SNPgenotype(s) to participate in a clinical trial of another type of drugthat may benefit them), etc. The SNP genotyping methods of the inventioncan also be useful for evaluating an individual's risk for developingCVD (particularly CHD, such as MI) and for predicting the likelihoodthat an individual who has previously had CVD will have a recurrence ofCVD again in the future (e.g., recurrent MI).

Nucleic acid samples can be genotyped to determine which allele(s)is/are present at any given genetic region (e.g., SNP position) ofinterest by methods well known in the art. The neighboring sequence canbe used to design SNP detection reagents such as oligonucleotide probes,which may optionally be implemented in a kit format. Exemplary SNPgenotyping methods are described in Chen et al., “Single nucleotidepolymorphism genotyping: biochemistry, protocol, cost and throughput,”Pharmacogenomics J 3(2):77-96 (2003); Kwok et al., “Detection of singlenucleotide polymorphisms,” Curr Issues Mol Biol 5(2):43-60 (April 2003);Shi, “Technologies for individual genotyping: detection of geneticpolymorphisms in drug targets and disease genes,” Am J Pharmacogenomics2(3):197-205 (2002); and Kwok, “Methods for genotyping single nucleotidepolymorphisms,” Annu Rev Genomics Hum Genet 2:235-58 (2001). Exemplarytechniques for high-throughput SNP genotyping are described inMarnellos, “High-throughput SNP analysis for genetic associationstudies,” Curr Opin Drug Discov Devel 6(3):317-21 (May 2003). Common SNPgenotyping methods include, but are not limited to, TaqMan assays,molecular beacon assays, nucleic acid arrays, allele-specific primerextension, allele-specific PCR, arrayed primer extension, homogeneousprimer extension assays, primer extension with detection by massspectrometry, pyrosequencing, multiplex primer extension sorted ongenetic arrays, ligation with rolling circle amplification, homogeneousligation, OLA (U.S. Pat. No. 4,988,167), multiplex ligation reactionsorted on genetic arrays, restriction-fragment length polymorphism,single base extension-tag assays, and the Invader assay. Such methodsmay be used in combination with detection mechanisms such as, forexample, luminescence or chemiluminescence detection, fluorescencedetection, time-resolved fluorescence detection, fluorescence resonanceenergy transfer, fluorescence polarization, mass spectrometry, andelectrical detection.

Various methods for detecting polymorphisms include, but are not limitedto, methods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science230:1242 (1985); Cotton et al., PNAS 85:4397 (1988); and Saleeba et al.,Meth. Enzymol 217:286-295 (1992)), comparison of the electrophoreticmobility of variant and wild type nucleic acid molecules (Orita et al.,PNAS 86:2766 (1989); Cotton et al., Mutat Res 285:125-144 (1993); andHayashi et al., Genet Anal Tech Appl 9:73-79 (1992)), and assaying themovement of polymorphic or wild-type fragments in polyacrylamide gelscontaining a gradient of denaturant using denaturing gradient gelelectrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)). Sequencevariations at specific locations can also be assessed by nucleaseprotection assays such as RNase and 51 protection or chemical cleavagemethods.

In a preferred embodiment, SNP genotyping is performed using the TaqManassay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos.5,210,015 and 5,538,848). The TaqMan assay detects the accumulation of aspecific amplified product during PCR. The TaqMan assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively, or vice versa. Alternatively, the reporter dye maybe at the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

Preferred TaqMan primer and probe sequences can readily be determinedusing the SNP and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, CA), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the SNPs of the present inventionare useful in, for example, screening individuals for their likelihoodof responding to statin treatment (i.e., benefiting from statintreatment), particularly individuals who have or are susceptible to CVD(particularly CHD, such as MI), or in screening for individuals who aresusceptible to developing CVD. These probes and primers can be readilyincorporated into a kit format. The present invention also includesmodifications of the Taqman assay well known in the art such as the useof Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) andother variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).

Another preferred method for genotyping the SNPs of the presentinvention is the use of two oligonucleotide probes in an OLA (see, e.g.,U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to asegment of a target nucleic acid with its 3′ most end aligned with theSNP site. A second probe hybridizes to an adjacent segment of the targetnucleic acid molecule directly 3′ to the first probe. The two juxtaposedprobes hybridize to the target nucleic acid molecule, and are ligated inthe presence of a linking agent such as a ligase if there is perfectcomplementarity between the 3′ most nucleotide of the first probe withthe SNP site. If there is a mismatch, ligation would not occur. Afterthe reaction, the ligated probes are separated from the target nucleicacid molecule, and detected as indicators of the presence of a SNP.

The following patents, patent applications, and published internationalpatent applications, which are all hereby incorporated by reference,provide additional information pertaining to techniques for carrying outvarious types of OLA. The following U.S. patents describe OLA strategiesfor performing SNP detection: U.S. Pat. Nos. 6,027,889; 6,268,148;5,494,810; 5,830,711 and 6,054,564. WO 97/31256 and WO 00/56927 describeOLA strategies for performing SNP detection using universal arrays,wherein a zipcode sequence can be introduced into one of thehybridization probes, and the resulting product, or amplified product,hybridized to a universal zip code array. U.S. application US01/17329(and U.S. Ser. No. 09/584,905) describes OLA (or LDR) followed by PCR,wherein zipcodes are incorporated into OLA probes, and amplified PCRproducts are determined by electrophoretic or universal zipcode arrayreadout. U.S. applications 60/427,818, 60/445,636, and 60/445,494describe SNPlex methods and software for multiplexed SNP detection usingOLA followed by PCR, wherein zipcodes are incorporated into OLA probes,and amplified PCR products are hybridized with a zipchute reagent, andthe identity of the SNP determined from electrophoretic readout of thezipchute. In some embodiments, OLA is carried out prior to PCR (oranother method of nucleic acid amplification). In other embodiments, PCR(or another method of nucleic acid amplification) is carried out priorto OLA.

Another method for SNP genotyping is based on mass spectrometry. Massspectrometry takes advantage of the unique mass of each of the fournucleotides of DNA. SNPs can be unambiguously genotyped by massspectrometry by measuring the differences in the mass of nucleic acidshaving alternative SNP alleles. MALDI-TOF (Matrix Assisted LaserDesorption Ionization-Time of Flight) mass spectrometry technology ispreferred for extremely precise determinations of molecular mass, suchas SNPs. Numerous approaches to SNP analysis have been developed basedon mass spectrometry. Preferred mass spectrometry-based methods of SNPgenotyping include primer extension assays, which can also be utilizedin combination with other approaches, such as traditional gel-basedformats and microarrays.

Typically, the primer extension assay involves designing and annealing aprimer to a template PCR amplicon upstream (5′) from a target SNPposition. A mix of dideoxynucleotide triphosphates (ddNTPs) and/ordeoxynucleotide triphosphates (dNTPs) are added to a reaction mixturecontaining template (e.g., a SNP-containing nucleic acid molecule whichhas typically been amplified, such as by PCR), primer, and DNApolymerase. Extension of the primer terminates at the first position inthe template where a nucleotide complementary to one of the ddNTPs inthe mix occurs. The primer can be either immediately adjacent (i.e., thenucleotide at the 3′ end of the primer hybridizes to the nucleotide nextto the target SNP site) or two or more nucleotides removed from the SNPposition. If the primer is several nucleotides removed from the targetSNP position, the only limitation is that the template sequence betweenthe 3′ end of the primer and the SNP position cannot contain anucleotide of the same type as the one to be detected, or this willcause premature termination of the extension primer. Alternatively, ifall four ddNTPs alone, with no dNTPs, are added to the reaction mixture,the primer will always be extended by only one nucleotide, correspondingto the target SNP position. In this instance, primers are designed tobind one nucleotide upstream from the SNP position (i.e., the nucleotideat the 3′ end of the primer hybridizes to the nucleotide that isimmediately adjacent to the target SNP site on the 5′ side of the targetSNP site). Extension by only one nucleotide is preferable, as itminimizes the overall mass of the extended primer, thereby increasingthe resolution of mass differences between alternative SNP nucleotides.Furthermore, mass-tagged ddNTPs can be employed in the primer extensionreactions in place of unmodified ddNTPs. This increases the massdifference between primers extended with these ddNTPs, thereby providingincreased sensitivity and accuracy, and is particularly useful fortyping heterozygous base positions. Mass-tagging also alleviates theneed for intensive sample-preparation procedures and decreases thenecessary resolving power of the mass spectrometer.

The extended primers can then be purified and analyzed by MALDI-TOF massspectrometry to determine the identity of the nucleotide present at thetarget SNP position. In one method of analysis, the products from theprimer extension reaction are combined with light absorbing crystalsthat form a matrix. The matrix is then hit with an energy source such asa laser to ionize and desorb the nucleic acid molecules into thegas-phase. The ionized molecules are then ejected into a flight tube andaccelerated down the tube towards a detector. The time between theionization event, such as a laser pulse, and collision of the moleculewith the detector is the time of flight of that molecule. The time offlight is precisely correlated with the mass-to-charge ratio (m/z) ofthe ionized molecule. Ions with smaller m/z travel down the tube fasterthan ions with larger m/z and therefore the lighter ions reach thedetector before the heavier ions. The time-of-flight is then convertedinto a corresponding, and highly precise, m/z. In this manner, SNPs canbe identified based on the slight differences in mass, and thecorresponding time of flight differences, inherent in nucleic acidmolecules having different nucleotides at a single base position. Forfurther information regarding the use of primer extension assays inconjunction with MALDI-TOF mass spectrometry for SNP genotyping, see,e.g., Wise et al., “A standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry,” Rapid CommunMass Spectrom 17(11):1195-202 (2003).

The following references provide further information describing massspectrometry-based methods for SNP genotyping: Bocker, “SNP and mutationdiscovery using base-specific cleavage and MALDI-TOF mass spectrometry,”Bioinformatics 19 Suppl 1:144-153 (July 2003); Storm et al., “MALDI-TOFmass spectrometry-based SNP genotyping,” Methods Mol Biol 212:241-62(2003); Jurinke et al., “The use of Mass ARRAY technology for highthroughput genotyping,” Adv Biochem Eng Biotechnol 77:57-74 (2002); andJurinke et al., “Automated genotyping using the DNA MassArraytechnology,” Methods Mol Biol 187:179-92 (2002).

SNPs can also be scored by direct DNA sequencing. A variety of automatedsequencing procedures can be utilized (e.g. Biotechniques 19:448(1995)), including sequencing by mass spectrometry. See, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al., Adv Chromatogr36:127-162 (1996); and Griffin et al., Appl Biochem Biotechnol38:147-159 (1993). The nucleic acid sequences of the present inventionenable one of ordinary skill in the art to readily design sequencingprimers for such automated sequencing procedures. Commercialinstrumentation, such as the Applied Biosystems 377, 3100, 3700, 3730,and 3730xl DNA Analyzers (Foster City, CA), is commonly used in the artfor automated sequencing.

Other methods that can be used to genotype the SNPs of the presentinvention include single-strand conformational polymorphism (SSCP), anddenaturing gradient gel electrophoresis (DGGE). Myers et al., Nature313:495 (1985). SSCP identifies base differences by alteration inelectrophoretic migration of single stranded PCR products, as describedin Orita et al., Proc. Nat. Acad. Single-stranded PCR products can begenerated by heating or otherwise denaturing double stranded PCRproducts. Single-stranded nucleic acids may refold or form secondarystructures that are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts are related to base-sequence differences at SNP positions. DGGEdifferentiates SNP alleles based on the different sequence-dependentstabilities and melting properties inherent in polymorphic DNA and thecorresponding differences in electrophoretic migration patterns in adenaturing gradient gel. PCR Technology: Principles and Applications forDNA Amplification Chapter 7, Erlich, ed., W.H. Freeman and Co, N.Y.(1992).

Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be usedto score SNPs based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. If the SNP affects a restriction enzyme cleavagesite, the SNP can be identified by alterations in restriction enzymedigestion patterns, and the corresponding changes in nucleic acidfragment lengths determined by gel electrophoresis.

SNP genotyping can include the steps of, for example, collecting abiological sample from a human subject (e.g., sample of tissues, cells,fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA,mRNA or both) from the cells of the sample, contacting the nucleic acidswith one or more primers which specifically hybridize to a region of theisolated nucleic acid containing a target SNP under conditions such thathybridization and amplification of the target nucleic acid regionoccurs, and determining the nucleotide present at the SNP position ofinterest, or, in some assays, detecting the presence or absence of anamplification product (assays can be designed so that hybridizationand/or amplification will only occur if a particular SNP allele ispresent or absent). In some assays, the size of the amplificationproduct is detected and compared to the length of a control sample; forexample, deletions and insertions can be detected by a change in size ofthe amplified product compared to a normal genotype.

SNP genotyping is useful for numerous practical applications, asdescribed below. Examples of such applications include, but are notlimited to, SNP-disease association analysis, disease predispositionscreening, disease diagnosis, disease prognosis, disease progressionmonitoring, determining therapeutic strategies based on an individual'sgenotype (“pharmacogenomics”), developing therapeutic agents based onSNP genotypes associated with a disease or likelihood of responding to adrug, stratifying patient populations for clinical trials of atherapeutic, preventive, or diagnostic agent, and predicting thelikelihood that an individual will experience toxic side effects from atherapeutic agent.

Analysis of Genetic Associations Between SNPs and Phenotypic Traits

SNP genotyping for disease diagnosis, disease predisposition screening,disease prognosis, determining drug responsiveness (pharmacogenomics),drug toxicity screening, and other uses described herein, typicallyrelies on initially establishing a genetic association between one ormore specific SNPs and the particular phenotypic traits of interest.

Different study designs may be used for genetic association studies.Modern Epidemiology 609-622, Lippincott, Williams & Wilkins (1998).Observational studies are most frequently carried out in which theresponse of the patients is not interfered with. The first type ofobservational study identifies a sample of persons in whom the suspectedcause of the disease is present and another sample of persons in whomthe suspected cause is absent, and then the frequency of development ofdisease in the two samples is compared. These sampled populations arecalled cohorts, and the study is a prospective study. The other type ofobservational study is case-control or a retrospective study. In typicalcase-control studies, samples are collected from individuals with thephenotype of interest (cases) such as certain manifestations of adisease, and from individuals without the phenotype (controls) in apopulation (target population) that conclusions are to be drawn from.Then the possible causes of the disease are investigatedretrospectively. As the time and costs of collecting samples incase-control studies are considerably less than those for prospectivestudies, case-control studies are the more commonly used study design ingenetic association studies, at least during the exploration anddiscovery stage.

Case-only studies are an alternative to case-control studies whengene-environment interaction is the association of interest (Piegorschet al., “Non-hierarchical logistic models and case-only designs forassessing susceptibility in population-based case-control studies”,Statistics in Medicine 13 (1994) pp 153-162). In a typical case-onlystudy of gene-environment interaction, genotypes are obtained only fromcases who are often selected from an existing cohort study. Theassociation between genotypes and the environmental factor is thenassessed and a significant association implies that the effect of theenvironmental factor on the endpoint of interest (the case definition)differs by genotype. The primary assumption underlying the test ofassociation in case-only studies is that the environmental effect ofinterest is independent of genotype (e.g., allocation to statin therapyis independent of genotype) and it has been shown that the case-onlydesign has more power than the case-control design to detectgene-environment interaction when this assumption is true in thepopulation (Yang et al., “Sample Size Requirements in Case-Only Designsto Detect Gene-Environment Interaction”, American Journal ofEpidemiology 146:9 (1997) pp 713-720). Selecting cases from a randomizedclinical trial may be an ideal setting in which to perform a case-onlystudy since genotypes will be independent of treatment by design.

In observational studies, there may be potential confounding factorsthat should be taken into consideration. Confounding factors are thosethat are associated with both the real cause(s) of the disease and thedisease itself, and they include demographic information such as age,gender, ethnicity as well as environmental factors. When confoundingfactors are not matched in cases and controls in a study, and are notcontrolled properly, spurious association results can arise. Ifpotential confounding factors are identified, they should be controlledfor by analysis methods explained below.

In a genetic association study, the cause of interest to be tested is acertain allele or a SNP or a combination of alleles or a haplotype fromseveral SNPs. Thus, tissue specimens (e.g., whole blood) from thesampled individuals may be collected and genomic DNA genotyped for theSNP(s) of interest. In addition to the phenotypic trait of interest,other information such as demographic (e.g., age, gender, ethnicity,etc.), clinical, and environmental information that may influence theoutcome of the trait can be collected to further characterize and definethe sample set. In many cases, these factors are known to be associatedwith diseases and/or SNP allele frequencies. There are likelygene-environment and/or gene-gene interactions as well. Analysis methodsto address gene-environment and gene-gene interactions (for example, theeffects of the presence of both susceptibility alleles at two differentgenes can be greater than the effects of the individual alleles at twogenes combined) are discussed below.

After all the relevant phenotypic and genotypic information has beenobtained, statistical analyses are carried out to determine if there isany significant correlation between the presence of an allele or agenotype with the phenotypic characteristics of an individual.Preferably, data inspection and cleaning are first performed beforecarrying out statistical tests for genetic association. Epidemiologicaland clinical data of the samples can be summarized by descriptivestatistics with tables and graphs. Data validation is preferablyperformed to check for data completion, inconsistent entries, andoutliers. Chi-squared tests and t-tests (Wilcoxon rank-sum tests ifdistributions are not normal) may then be used to check for significantdifferences between cases and controls for discrete and continuousvariables, respectively. To ensure genotyping quality, Hardy-Weinbergdisequilibrium tests can be performed on cases and controls separately.Significant deviation from Hardy-Weinberg equilibrium (HWE) in bothcases and controls for individual markers can be indicative ofgenotyping errors. If HWE is violated in a majority of markers, it isindicative of population substructure that should be furtherinvestigated. Moreover, Hardy-Weinberg disequilibrium in cases only canindicate genetic association of the markers with the disease. B. Weir,Genetic Data Analysis, Sinauer (1990).

To test whether an allele of a single SNP is associated with the case orcontrol status of a phenotypic trait, one skilled in the art can compareallele frequencies in cases and controls. Standard chi-squared tests andFisher exact tests can be carried out on a 2×2 table (2 SNP alleles×2outcomes in the categorical trait of interest). To test whethergenotypes of a SNP are associated, chi-squared tests can be carried outon a 3×2 table (3 genotypes×2 outcomes). Score tests are also carriedout for genotypic association to contrast the three genotypicfrequencies (major homozygotes, heterozygotes and minor homozygotes) incases and controls, and to look for trends using 3 different modes ofinheritance, namely dominant (with contrast coefficients 2, −1, −1),additive or allelic (with contrast coefficients 1, 0, −1) and recessive(with contrast coefficients 1, 1, −2). Odds ratios for minor versusmajor alleles, and odds ratios for heterozygote and homozygote variantsversus the wild type genotypes are calculated with the desiredconfidence limits, usually 95%.

In order to control for confounders and to test for interaction andeffect modifiers, stratified analyses may be performed using stratifiedfactors that are likely to be confounding, including demographicinformation such as age, ethnicity, and gender, or an interactingelement or effect modifier, such as a known major gene (e.g., APOE forAlzheimer's disease or HLA genes for autoimmune diseases), orenvironmental factors such as smoking in lung cancer. Stratifiedassociation tests may be carried out using Cochran-Mantel-Haenszel teststhat take into account the ordinal nature of genotypes with 0, 1, and 2variant alleles. Exact tests by StatXact may also be performed whencomputationally possible. Another way to adjust for confounding effectsand test for interactions is to perform stepwise multiple logisticregression analysis using statistical packages such as SAS or R.Logistic regression is a model-building technique in which the bestfitting and most parsimonious model is built to describe the relationbetween the dichotomous outcome (for instance, getting a certain diseaseor not) and a set of independent variables (for instance, genotypes ofdifferent associated genes, and the associated demographic andenvironmental factors). The most common model is one in which the logittransformation of the odds ratios is expressed as a linear combinationof the variables (main effects) and their cross-product terms(interactions). Hosmer and Lemeshow, Applied Logistic Regression, Wiley(2000). To test whether a certain variable or interaction issignificantly associated with the outcome, coefficients in the model arefirst estimated and then tested for statistical significance of theirdeparture from zero.

In addition to performing association tests one marker at a time,haplotype association analysis may also be performed to study a numberof markers that are closely linked together. Haplotype association testscan have better power than genotypic or allelic association tests whenthe tested markers are not the disease-causing mutations themselves butare in linkage disequilibrium with such mutations. The test will even bemore powerful if the disease is indeed caused by a combination ofalleles on a haplotype (e.g., APOE is a haplotype formed by 2 SNPs thatare very close to each other). In order to perform haplotype associationeffectively, marker-marker linkage disequilibrium measures, both D′ andr², are typically calculated for the markers within a gene to elucidatethe haplotype structure. Recent studies in linkage disequilibriumindicate that SNPs within a gene are organized in block pattern, and ahigh degree of linkage disequilibrium exists within blocks and verylittle linkage disequilibrium exists between blocks. Daly et al, NatureGenetics 29:232-235 (2001). Haplotype association with the diseasestatus can be performed using such blocks once they have beenelucidated.

Haplotype association tests can be carried out in a similar fashion asthe allelic and genotypic association tests. Each haplotype in a gene isanalogous to an allele in a multi-allelic marker. One skilled in the artcan either compare the haplotype frequencies in cases and controls ortest genetic association with different pairs of haplotypes. It has beenproposed that score tests can be done on haplotypes using the program“haplo.score.” Schaid et al, Am J Hum Genet 70:425-434 (2002). In thatmethod, haplotypes are first inferred by EM algorithm and score testsare carried out with a generalized linear model (GLM) framework thatallows the adjustment of other factors.

An important decision in the performance of genetic association tests isthe determination of the significance level at which significantassociation can be declared when the P value of the tests reaches thatlevel. In an exploratory analysis where positive hits will be followedup in subsequent confirmatory testing, an unadjusted P value <0.2 (asignificance level on the lenient side), for example, may be used forgenerating hypotheses for significant association of a SNP with certainphenotypic characteristics of a disease. It is preferred that a p-value<0.05 (a significance level traditionally used in the art) is achievedin order for a SNP to be considered to have an association with adisease. It is more preferred that a p-value <0.01 (a significance levelon the stringent side) is achieved for an association to be declared.When hits are followed up in confirmatory analyses in more samples ofthe same source or in different samples from different sources,adjustment for multiple testing will be performed as to avoid excessnumber of hits while maintaining the experiment-wide error rates at0.05. While there are different methods to adjust for multiple testingto control for different kinds of error rates, a commonly used butrather conservative method is Bonferroni correction to control theexperiment-wise or family-wise error rate. Westfall et al., Multiplecomparisons and multiple tests, SAS Institute (1999). Permutation teststo control for the false discovery rates, FDR, can be more powerful.Benjamini and Hochberg, Journal of the Royal Statistical Society, SeriesB 57:1289-1300 (1995); Westfall and Young, Resampling-based MultipleTesting, Wiley (1993). Such methods to control for multiplicity would bepreferred when the tests are dependent and controlling for falsediscovery rates is sufficient as opposed to controlling for theexperiment-wise error rates.

In replication studies using samples from different populations afterstatistically significant markers have been identified in theexploratory stage, meta-analyses can then be performed by combiningevidence of different studies. Modern Epidemiology 643-673, Lippincott,Williams & Wilkins (1998). If available, association results known inthe art for the same SNPs can be included in the meta-analyses.

Since both genotyping and disease status classification can involveerrors, sensitivity analyses may be performed to see how odds ratios andp-values would change upon various estimates on genotyping and diseaseclassification error rates.

It has been well known that subpopulation-based sampling bias betweencases and controls can lead to spurious results in case-controlassociation studies when prevalence of the disease is associated withdifferent subpopulation groups. Ewens and Spielman, Am J Hum Genet62:450-458 (1995). Such bias can also lead to a loss of statisticalpower in genetic association studies. To detect populationstratification, Pritchard and Rosenberg suggested typing markers thatare unlinked to the disease and using results of association tests onthose markers to determine whether there is any populationstratification. Pritchard et al., Am J Hum Gen 65:220-228 (1999). Whenstratification is detected, the genomic control (GC) method as proposedby Devlin and Roeder can be used to adjust for the inflation of teststatistics due to population stratification. Devlin et al., Biometrics55:997-1004 (1999). The GC method is robust to changes in populationstructure levels as well as being applicable to DNA pooling designs.Devlin et al., Genet Epidem 21:273-284 (2001).

While Pritchard's method recommended using 15-20 unlinked microsatellitemarkers, it suggested using more than 30 biallelic markers to get enoughpower to detect population stratification. For the GC method, it hasbeen shown that about 60-70 biallelic markers are sufficient to estimatethe inflation factor for the test statistics due to populationstratification. Bacanu et al., Am J Hum Genet 66:1933-1944 (2000).Hence, 70 intergenic SNPs can be chosen in unlinked regions as indicatedin a genome scan. Kehoe et al., Hum Mol Genet 8:237-245 (1999).

Once individual risk factors, genetic or non-genetic, have been foundfor the predisposition to disease, the next step is to set up aclassification/prediction scheme to predict the category (for instance,disease or no-disease) that an individual will be in depending on hisgenotypes of associated SNPs and other non-genetic risk factors.Logistic regression for discrete trait and linear regression forcontinuous trait are standard techniques for such tasks. Draper andSmith, Applied Regression Analysis, Wiley (1998). Moreover, othertechniques can also be used for setting up classification. Suchtechniques include, but are not limited to, MART, CART, neural network,and discriminant analyses that are suitable for use in comparing theperformance of different methods. The Elements of Statistical Learning,Hastie, Tibshirani & Friedman, Springer (2002).

For further information about genetic association studies, see Balding,“A tutorial on statistical methods for population association studies”,Nature Reviews Genetics 7, 781 (2006).

Disease Diagnosis and Predisposition Screening

Information on association/correlation between genotypes anddisease-related phenotypes can be exploited in several ways. Forexample, in the case of a highly statistically significant associationbetween one or more SNPs with predisposition to a disease for whichtreatment is available, detection of such a genotype pattern in anindividual may justify immediate administration of treatment, or atleast the institution of regular monitoring of the individual. Detectionof the susceptibility alleles associated with serious disease in acouple contemplating having children may also be valuable to the couplein their reproductive decisions. In the case of a weaker but stillstatistically significant association between a SNP and a human disease,immediate therapeutic intervention or monitoring may not be justifiedafter detecting the susceptibility allele or SNP. Nevertheless, thesubject can be motivated to begin simple life-style changes (e.g., diet,exercise) that can be accomplished at little or no cost to theindividual but would confer potential benefits in reducing the risk ofdeveloping conditions for which that individual may have an increasedrisk by virtue of having the risk allele(s).

The SNPs of the invention may contribute to responsiveness of anindividual to statin treatment, or to the development of CVD (e.g., CHD,such as MI), in different ways. Some polymorphisms occur within aprotein coding sequence and contribute to disease phenotype by affectingprotein structure. Other polymorphisms occur in noncoding regions butmay exert phenotypic effects indirectly via influence on, for example,replication, transcription, and/or translation. A single SNP may affectmore than one phenotypic trait. Likewise, a single phenotypic trait maybe affected by multiple SNPs in different genes.

As used herein, the terms “diagnose,” “diagnosis,” and “diagnostics”include, but are not limited to, any of the following: detection of CVD(e.g., CHD, such as MI) that an individual may presently have,predisposition/susceptibility/predictive screening (i.e., determiningwhether an individual has an increased or decreased risk of developingCVD in the future), predicting recurrence of CVD (e.g., recurrent MI) inan individual, determining a particular type or subclass of CVD in anindividual who currently or previously had CVD, confirming orreinforcing a previously made diagnosis of CVD, evaluating anindividual's likelihood of responding positively to a particulartreatment or therapeutic agent (i.e., benefiting) such as statintreatment (particularly treatment or prevention of CVD, especially CHDsuch as MI, using statins), determining or selecting a therapeutic orpreventive strategy that an individual is most likely to positivelyrespond to (e.g., selecting a particular therapeutic agent such as astatin, or combination of therapeutic agents, or selecting a particularstatin from among other statins, or determining a dosing regimen orselecting a dosage formulation, etc.), classifying (orconfirming/reinforcing) an individual as a responder/non-responder (ordetermining a particular subtype of responder/non-responder) withrespect to the individual's response to a drug treatment such as statintreatment, and predicting whether a patient is likely to experiencetoxic effects from a particular treatment or therapeutic compound. Suchdiagnostic uses can be based on the SNPs individually or a uniquecombination or SNPs disclosed herein, as well as SNP haplotypes.

Haplotypes are particularly useful in that, for example, fewer SNPs canbe genotyped to determine if a particular genomic region harbors a locusthat influences a particular phenotype, such as in linkagedisequilibrium-based SNP association analysis.

Linkage disequilibrium (LD) refers to the co-inheritance of alleles(e.g., alternative nucleotides) at two or more different SNP sites atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given population. The expectedfrequency of co-occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage equilibrium.” In contrast, LDrefers to any non-random genetic association between allele(s) at two ormore different SNP sites, which is generally due to the physicalproximity of the two loci along a chromosome. LD can occur when two ormore SNPs sites are in close physical proximity to each other on a givenchromosome and therefore alleles at these SNP sites will tend to remainunseparated for multiple generations with the consequence that aparticular nucleotide (allele) at one SNP site will show a non-randomassociation with a particular nucleotide (allele) at a different SNPsite located nearby. Hence, genotyping one of the SNP sites will givealmost the same information as genotyping the other SNP site that is inLD.

Various degrees of LD can be encountered between two or more SNPs withthe result being that some SNPs are more closely associated (i.e., instronger LD) than others. Furthermore, the physical distance over whichLD extends along a chromosome differs between different regions of thegenome, and therefore the degree of physical separation between two ormore SNP sites necessary for LD to occur can differ between differentregions of the genome.

For diagnostic purposes and similar uses, if a particular SNP site isfound to be useful for, for example, predicting an individual's responseto statin treatment or an individual's susceptibility to CVD, then theskilled artisan would recognize that other SNP sites which are in LDwith this SNP site would also be useful for the same purposes. Thus,polymorphisms (e.g., SNPs and/or haplotypes) that are not the actualdisease-causing (causative) polymorphisms, but are in LD with suchcausative polymorphisms, are also useful. In such instances, thegenotype of the polymorphism(s) that is/are in LD with the causativepolymorphism is predictive of the genotype of the causative polymorphismand, consequently, predictive of the phenotype (e.g., response to statintreatment or risk for developing CVD) that is influenced by thecausative SNP(s). Therefore, polymorphic markers that are in LD withcausative polymorphisms are useful as diagnostic markers, and areparticularly useful when the actual causative polymorphism(s) is/areunknown.

Examples of polymorphisms that can be in LD with one or more causativepolymorphisms (and/or in LD with one or more polymorphisms that have asignificant statistical association with a condition) and thereforeuseful for diagnosing the same condition that the causative/associatedSNP(s) is used to diagnose, include other SNPs in the same gene,protein-coding, or mRNA transcript-coding region as thecausative/associated SNP, other SNPs in the same exon or same intron asthe causative/associated SNP, other SNPs in the same haplotype block asthe causative/associated SNP, other SNPs in the same intergenic regionas the causative/associated SNP, SNPs that are outside but near a gene(e.g., within 6 kb on either side, 5′ or 3′, of a gene boundary) thatharbors a causative/associated SNP, etc. Such useful LD SNPs can beselected from among the SNPs disclosed in Table 3, for example.

Linkage disequilibrium in the human genome is reviewed in Wall et al.,“Haplotype blocks and linkage disequilibrium in the human genome,” NatRev Genet 4(8):587-97 (August 2003); Garner et al., “On selectingmarkers for association studies: patterns of linkage disequilibriumbetween two and three diallelic loci,” Genet Epidemiol 24(1):57-67(January 2003); Ardlie et al., “Patterns of linkage disequilibrium inthe human genome,” Nat Rev Genet 3(4):299-309 (April 2002); erratum inNat Rev Genet 3(7):566 (July 2002); and Remm et al., “High-densitygenotyping and linkage disequilibrium in the human genome usingchromosome 22 as a model,” Curr Opin Chem Biol 6(1):24-30 (February2002); J. B. S. Haldane, “The combination of linkage values, and thecalculation of distances between the loci of linked factors,” J Genet8:299-309 (1919); G. Mendel, Versuche über Pflanzen-Hybriden.Verhandlungen des naturforschenden Vereines in Brünn (Proceedings of theNatural History Society of Brünn) (1866); Genes IV, B. Lewin, ed.,Oxford University Press, N.Y. (1990); D. L. Hartl and A. G. ClarkPrinciples of Population Genetics 2^(nd) ed., Sinauer Associates, Inc.,Mass. (1989); J. H. Gillespie Population Genetics: A Concise Guide.2^(nd) ed., Johns Hopkins University Press (2004); R. C. Lewontin, “Theinteraction of selection and linkage. I. General considerations;heterotic models,” Genetics 49:49-67 (1964); P. G. Hoel, Introduction toMathematical Statistics 2^(nd) ed., John Wiley & Sons, Inc., N. Y.(1954); R. R. Hudson, “Two-locus sampling distributions and theirapplication,” Genetics 159:1805-1817 (2001); A. P. Dempster, N. M.Laird, D. B. Rubin, “Maximum likelihood from incomplete data via the EMalgorithm,” J R Stat Soc 39:1-38 (1977); L. Excoffier, M. Slatkin,“Maximum-likelihood estimation of molecular haplotype frequencies in adiploid population,” Mol Biol Evol 12(5):921-927 (1995); D. A. Tregouet,S. Escolano, L. Tiret, A. Mallet, J. L. Golmard, “A new algorithm forhaplotype-based association analysis: the Stochastic-EM algorithm,” AnnHum Genet 68(Pt 2):165-177 (2004); A. D. Long and C. H. Langley C H,“The power of association studies to detect the contribution ofcandidate genetic loci to variation in complex traits,” Genome Research9:720-731 (1999); A. Agresti, Categorical Data Analysis, John Wiley &Sons, Inc., N. Y. (1990); K. Lange, Mathematical and Statistical Methodsfor Genetic Analysis, Springer-Verlag New York, Inc., N. Y. (1997); TheInternational HapMap Consortium, “The International HapMap Project,”Nature 426:789-796 (2003); The International HapMap Consortium, “Ahaplotype map of the human genome,” Nature 437:1299-1320 (2005); G. A.Thorisson, A. V. Smith, L. Krishnan, L. D. Stein, “The InternationalHapMap Project Web Site,” Genome Research 15:1591-1593 (2005); G.McVean, C. C. A. Spencer, R. Chaix, “Perspectives on human geneticvariation from the HapMap project,” PLoS Genetics 1(4):413-418 (2005);J. N. Hirschhorn, M. J. Daly, “Genome-wide association studies forcommon diseases and complex traits,” Nat Genet 6:95-108 (2005); S. J.Schrodi, “A probabilistic approach to large-scale association scans: asemi-Bayesian method to detect disease-predisposing alleles,” SAGMB4(1):31 (2005); W. Y. S. Wang, B. J. Barratt, D. G. Clayton, J. A. Todd,“Genome-wide association studies: theoretical and practical concerns,”Nat Rev Genet 6:109-118 (2005); J. K. Pritchard, M. Przeworski, “Linkagedisequilibrium in humans: models and data,” Am J Hum Genet 69:1-14(2001).

As discussed above, an aspect of the present invention relates to SNPsthat are in LD with an interrogated SNP and which can also be used asvalid markers for determining an individual's likelihood of benefitingfrom statin treatment, or whether an individual has an increased ordecreased risk of having or developing CVD. As used herein, the term“interrogated SNP” refers to SNPs that have been found to be associatedwith statin response, particularly for reducing CVD risk, usinggenotyping results and analysis, or other appropriate experimentalmethod as exemplified in the working examples described in thisapplication. As used herein, the term “LD SNP” refers to a SNP that hasbeen characterized as a SNP associated with statin response or anincreased or decreased risk of CVD due to their being in LD with the“interrogated SNP” under the methods of calculation described in theapplication. Below, applicants describe the methods of calculation withwhich one of ordinary skilled in the art may determine if a particularSNP is in LD with an interrogated SNP. The parameter r² is commonly usedin the genetics art to characterize the extent of linkage disequilibriumbetween markers (Hudson, 2001). As used herein, the term “in LD with”refers to a particular SNP that is measured at above the threshold of aparameter such as r² with an interrogated SNP.

It is now common place to directly observe genetic variants in a sampleof chromosomes obtained from a population. Suppose one has genotype dataat two genetic markers located on the same chromosome, for the markers Aand B. Further suppose that two alleles segregate at each of these twomarkers such that alleles A₁ and A₂ can be found at marker A and allelesB₁ and B₂ at marker B. Also assume that these two markers are on a humanautosome. If one is to examine a specific individual and find that theyare heterozygous at both markers, such that their two-marker genotype isA₁A₂B₁B₂, then there are two possible configurations: the individual inquestion could have the alleles A₁B₁ on one chromosome and A₂B₂ on theremaining chromosome; alternatively, the individual could have allelesA₁B₂ on one chromosome and A₂B₁ on the other. The arrangement of alleleson a chromosome is called a haplotype. In this illustration, theindividual could have haplotypes A₁B₁/A₂B₂ or A₁B₂/A₂B₁ (see Hartl andClark (1989) for a more complete description). The concept of linkageequilibrium relates the frequency of haplotypes to the allelefrequencies.

Assume that a sample of individuals is selected from a largerpopulation. Considering the two markers described above, each having twoalleles, there are four possible haplotypes: A₁B₁, A₁B₂, A₂B₁ and A₂B₂.Denote the frequencies of these four haplotypes with the followingnotation.P ₁₁=freq(A ₁ B ₁)  (1)P ₁₂=freq(A ₁ B ₂  (2)P ₂₁=freq(A ₂ B ₁)  (3)P ₂₂=freq(A ₂ B ₂)  (4)The allele frequencies at the two markers are then the sum of differenthaplotype frequencies, it is straightforward to write down a similar setof equations relating single-marker allele frequencies to two-markerhaplotype frequencies:p ₁=freq(A ₁)=P ₁₁ +P ₁₂  (5)p ₂=freq(A ₂)=P ₂₁ +P ₂₂  (6)q ₁=freq(B ₁)=P ₁₁ +P ₂₁  (7)q ₂=freq(B ₂)=P ₁₂ +P ₂₂  (8)Note that the four haplotype frequencies and the allele frequencies ateach marker must sum to a frequency of 1.P ₁₁ +P ₁₂ +P ₂₁ +P ₂₂=1  (9)p ₁ +p ₂=1  (10)q ₁ +q ₂=1  (11)If there is no correlation between the alleles at the two markers, onewould expect that the frequency of the haplotypes would be approximatelythe product of the composite alleles. Therefore,P ₁₁ ≠p ₁ q ₁  (12)P ₁₂ ≠p ₁ q ₂  (13)P ₂₁ ≠p ₂ q ₁  (14)P ₂₂ ≠p ₂ q ₂  (15)These approximating equations (12)-(15) represent the concept of linkageequilibrium where there is independent assortment between the twomarkers—the alleles at the two markers occur together at random. Theseare represented as approximations because linkage equilibrium andlinkage disequilibrium are concepts typically thought of as propertiesof a sample of chromosomes; and as such they are susceptible tostochastic fluctuations due to the sampling process. Empirically, manypairs of genetic markers will be in linkage equilibrium, but certainlynot all pairs.

Having established the concept of linkage equilibrium above, applicantscan now describe the concept of linkage disequilibrium (LD), which isthe deviation from linkage equilibrium. Since the frequency of the A₁B₁haplotype is approximately the product of the allele frequencies for A₁and B₁ under the assumption of linkage equilibrium as statedmathematically in (12), a simple measure for the amount of departurefrom linkage equilibrium is the difference in these two quantities, D,D=P ₁₁ −p ₁ q ₁  (16)D=0 indicates perfect linkage equilibrium. Substantial departures fromD=0 indicates LD in the sample of chromosomes examined. Many propertiesof D are discussed in Lewontin (1964) including the maximum and minimumvalues that D can take. Mathematically, using basic algebra, it can beshown that D can also be written solely in terms of haplotypes:D=P ₁₁ P ₂₂ −P ₁₂ P ₂₁  (17)If one transforms D by squaring it and subsequently dividing by theproduct of the allele frequencies of A₁, A₂, B₁ and B₂, the resultingquantity, called r², is equivalent to the square of the Pearson'scorrelation coefficient commonly used in statistics (e.g., Hoel, 1954).

$\begin{matrix}{r^{2} = \frac{D^{2}}{p_{1}p_{2}q_{1}q_{2}}} & (18)\end{matrix}$

As with D, values of r² close to 0 indicate linkage equilibrium betweenthe two markers examined in the sample set. As values of r² increase,the two markers are said to be in linkage disequilibrium. The range ofvalues that r² can take are from 0 to 1. r²=1 when there is a perfectcorrelation between the alleles at the two markers.

In addition, the quantities discussed above are sample-specific. And assuch, it is necessary to formulate notation specific to the samplesstudied. In the approach discussed here, three types of samples are ofprimary interest: (i) a sample of chromosomes from individuals affectedby a disease-related phenotype (cases), (ii) a sample of chromosomesobtained from individuals not affected by the disease-related phenotype(controls), and (iii) a standard sample set used for the construction ofhaplotypes and calculation pairwise linkage disequilibrium. For theallele frequencies used in the development of the method describedbelow, an additional subscript will be added to denote either the caseor control sample sets.p _(1,cs)=freq(A ₁ in cases)  (19)p _(2,cs)=freq(A ₂ in cases)  (20)q _(1,cs)=freq(B ₁ in cases)  (21)q _(2,cs)=freq(B ₂ in cases)  (22)Similarly,p _(1,ct)=freq(A ₁ in controls)  (23)p _(2,ct)=freq(A ₂ in controls)  (24)q _(1,ct)=freq(B ₁ in controls)  (25)q _(2,ct)=freq(B ₂ in controls)  (26)

As a well-accepted sample set is necessary for robust linkagedisequilibrium calculations, data obtained from the International HapMapproject (The International HapMap Consortium 2003, 2005; Thoris son etal, 2005; McVean et al, 2005) can be used for the calculation ofpairwise r² values. Indeed, the samples genotyped for the InternationalHapMap Project were selected to be representative examples from varioushuman sub-populations with sufficient numbers of chromosomes examined todraw meaningful and robust conclusions from the patterns of geneticvariation observed. The International HapMap project website(hapmap.org) contains a description of the project, methods utilized andsamples examined. It is useful to examine empirical data to get a senseof the patterns present in such data.

Haplotype frequencies were explicit arguments in equation (18) above.However, knowing the 2-marker haplotype frequencies requires that phaseto be determined for doubly heterozygous samples. When phase is unknownin the data examined, various algorithms can be used to infer phase fromthe genotype data. This issue was discussed earlier where the doublyheterozygous individual with a 2-SNP genotype of A₁A₂B₁B₂ could have oneof two different sets of chromosomes: A₁B₁/A₂B₂ or A₁B₂/A₂B₁. One suchalgorithm to estimate haplotype frequencies is theexpectation-maximization (EM) algorithm first formalized by Dempster etal. (1977). This algorithm is often used in genetics to infer haplotypefrequencies from genotype data (e.g. Excoffier and Slatkin (1995);Tregouet et al. (2004)). It should be noted that for the two-SNP caseexplored here, EM algorithms have very little error provided that theallele frequencies and sample sizes are not too small. The impact on r²values is typically negligible.

As correlated genetic markers share information, interrogation of SNPmarkers in LD with a disease-associated SNP marker can also havesufficient power to detect disease association (Long and Langley(1999)). The relationship between the power to directly finddisease-associated alleles and the power to indirectly detectdisease-association was investigated by Pritchard and Przeworski (2001).In a straight-forward derivation, it can be shown that the power todetect disease association indirectly at a marker locus in linkagedisequilibrium with a disease-association locus is approximately thesame as the power to detect disease-association directly at thedisease-association locus if the sample size is increased by a factor of

$\frac{1}{r^{2}}$(the reciprocal of equation 18) at the marker in comparison with thedisease-association locus.

Therefore, if one calculated the power to detect disease-associationindirectly with an experiment having N samples, then equivalent power todirectly detect disease-association (at the actualdisease-susceptibility locus) would necessitate an experiment usingapproximately r²N samples. This elementary relationship between power,sample size and linkage disequilibrium can be used to derive an r²threshold value useful in determining whether or not genotyping markersin linkage disequilibrium with a SNP marker directly associated withdisease status has enough power to indirectly detectdisease-association.

To commence a derivation of the power to detect disease-associatedmarkers through an indirect process, define the effective chromosomalsample size as

$\begin{matrix}{{n = \frac{4N_{cs}N_{ct}}{N_{cs} + N_{ct}}};} & (27)\end{matrix}$where N_(cs) and N_(ct) are the numbers of diploid cases and controls,respectively. This is necessary to handle situations where the numbersof cases and controls are not equivalent. For equal case and controlsample sizes, N_(cs)=N_(ct)=N, the value of the effective number ofchromosomes is simply n=2N—as expected. Let power be calculated for asignificance level a (such that traditional P-values below α will bedeemed statistically significant). Define the standard Gaussiandistribution function as Φ(•). Mathematically,

$\begin{matrix}{{\Phi(x)} = {\frac{1}{\sqrt{2\pi}}{\int\limits_{- \infty}^{x}{e^{\frac{\theta^{2}}{2}}d\theta}}}} & (28)\end{matrix}$Alternatively, the following error function notation (Erf) may also beused,

$\begin{matrix}{{\Phi(x)} = {\frac{1}{2}\left\lbrack {1 + {{Erf}\ \left( \frac{x}{\sqrt{2}} \right)}} \right\rbrack}} & (29)\end{matrix}$

For example, Φ(1.644854)=0.95. The value of r² may be derived to yield apre-specified minimum amount of power to detect disease associationthough indirect interrogation. Noting that the LD SNP marker could bethe one that is carrying the disease-association allele, therefore thatthis approach constitutes a lower-bound model where all indirect powerresults are expected to be at least as large as those interrogated.

Denote by β the error rate for not detecting truly disease-associatedmarkers. Therefore, 1−β is the classical definition of statisticalpower. Substituting the Pritchard-Pzreworski result into the samplesize, the power to detect disease association at a significance level ofα is given by the approximation

$\begin{matrix}{{{1 - \beta} \cong {\Phi\left\lbrack {\frac{❘{q_{1,{cs}} - q_{1,{ct}}}❘}{\sqrt{\frac{{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}}{r^{2}n}}} - Z_{1 - \frac{\alpha}{2}}} \right\rbrack}};} & (30)\end{matrix}$where Z_(u) is the inverse of the standard normal cumulativedistribution evaluated at u (u∈(0,1)). Z_(u)=Φ⁻¹(u), where Φ(Φ⁻¹(u))=Φ⁻¹(Φ(u))=u. For example, setting α=0.05, and therefore1−α/2=0.975, one obtains Z_(0.975)=1.95996. Next, setting power equal toa threshold of a minimum power of T,

$\begin{matrix}{T = {\Phi\left\lbrack {\frac{❘{q_{1,{cs}} - q_{1,{ct}}}❘}{\sqrt{\frac{{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}}{r^{2}n}}} - Z_{1 - \frac{\alpha}{2}}} \right\rbrack}} & (31)\end{matrix}$and solving for r², the following threshold r² is obtained:

$\begin{matrix}{{r_{T}^{2} = {\frac{\left\lfloor {{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}} \right\rfloor}{{n\left( {q_{1,{cs}} - q_{1,{ct}}} \right)}^{2}}\left\lbrack {{\Phi^{- 1}(T)} + Z_{1 - \frac{\alpha}{2}}} \right\rbrack}^{2}}{{Or},}} & (32)\end{matrix}$ $\begin{matrix}{r_{T}^{2} = {\frac{\left( {Z_{T} + Z_{1 - \frac{\alpha}{2}}} \right)^{2}}{n}\left\lbrack \frac{q_{1,{cs}} - \left( q_{1,{cs}} \right)^{2} + q_{1,{ct}} - \left( q_{1,{ct}} \right)^{2}}{\left( {q_{1,{cs}} - q_{1,{ct}}} \right)^{2}} \right\rbrack}} & (33)\end{matrix}$

Suppose that r² is calculated between an interrogated SNP and a numberof other SNPs with varying levels of LD with the interrogated SNP. Thethreshold value r_(T) ² is the minimum value of linkage disequilibriumbetween the interrogated SNP and the potential LD SNPs such that the LDSNP still retains a power greater or equal to T for detectingdisease-association. For example, suppose that SNP rs200 is genotyped ina case-control disease-association study and it is found to beassociated with a disease phenotype. Further suppose that the minorallele frequency in 1,000 case chromosomes was found to be 16% incontrast with a minor allele frequency of 10% in 1,000 controlchromosomes. Given those measurements one could have predicted, prior tothe experiment, that the power to detect disease association at asignificance level of 0.05 was quite high—approximately 98% using a testof allelic association. Applying equation (32) one can calculate aminimum value of r² to indirectly assess disease association assumingthat the minor allele at SNP rs200 is truly disease-predisposing for athreshold level of power. If one sets the threshold level of power to be80%, then r_(T) ²=0.489 given the same significance level and chromosomenumbers as above. Hence, any SNP with a pairwise r² value with rs200greater than 0.489 is expected to have greater than 80% power to detectthe disease association. Further, this is assuming the conservativemodel where the LD SNP is disease-associated only through linkagedisequilibrium with the interrogated SNP rs200.

Imputation

Genotypes of SNPs can be imputed without actually having to be directlygenotyped (referred to as “imputation”), by using known haplotypeinformation. Imputation is a process to provide “missing” data, eithermissing individual genotypes (alleles) or missing SNPs and concomitantgenotypes, which have not been directly genotyped (i.e., assayed).Imputation is particularly useful for identifying disease associationsfor specific ungenotyped SNPs by inferring the missing genotypes tothese ungenotyped SNPs. Although the process uses similar information toLD, since the phasing and imputation process uses information frommultiple SNPs at the same time, the phased haplotype, it is able toinfer the genotype and achieve high identifiable accuracy. Genotypeinformation (such as from the HapMap project by The International HapMapConsortium, NCBI, NLM, NIH) can be used to infer haplotype phase andimpute genotypes for SNPs that are not directly genotyped in a givenindividual or sample set (such as for a disease association study). Ingeneral, imputation uses a reference dataset in which the genotypes ofpotential SNPs that are to be tested for disease association have beendetermined in multiple individuals (such as in HapMap); the individualsin the reference dataset are then haplotype phased. This phasing can bedone with independent programs such as fastPHASE (Sheet and Stephens, AmJ Hum Genet (2006) 76: 629-644) or a combination program such as BEAGLEwhich does both the phasing and the imputation. The reference phasedhaplotypes and process can be checked using the children of the HapMapindividual parents, among other mechanisms. Once the reference phasedhaplotypes have been created, the imputation of additional individualsfor SNPs genotyped or complete sets of SNPs that have not been directlygenotyped can then proceed. The HapMap dataset is particularly useful asthe reference dataset, however other datasets can be used. Since theimputation creates new concommitant phased haplotypes for individuals inthe association study and these contain other SNPs within the genomicregion, these ungenotyped but imputed SNPs can also be tested fordisease assocations (or other traits). Certain exemplary methods forhaplotype phase inference and imputation of missing genotypes utilizethe BEAGLE genetic analysis program, (Browning, Hum Genet (2008)124:439-450).

Thus, SNPs for which genotypes are imputed can be tested for associationwith a disease or other trait even though these SNPs are not directlygenotyped. The SNPs for which genotypes are imputed have genotype dataavailable in the reference dataset, e.g. HapMap individuals, but theyare not directly genotyped in a particular individual or sample set(such as in a particular disease association study).

In addition to using a reference dataset (e.g., HapMap) to imputegenotypes of SNPs that are not directly genotyped in a study, imputationcan provide genotypes of SNPs that were directly genotyped in a studybut for which the genotypes are missing in some or most of theindividuals for some reason, such as because they failed to pass qualitycontrol. Imputation can also be used to combine genotyping results frommultiple studies in which different sets of SNPs were genotyped toconstruct a complete meta-analysis. For example, genotyped and imputedgenotyped SNP results from multiple different studies can be combined,and the overlapping SNP genotypes (e.g., genotyped in one study, imputedin another study or imputed in both or genotyped in both) can beanalyzed across all of the studies (Browning, Hum Genet (2008)124:439-450).

For a review of imputation (as well as the BEAGLE program), seeBrowning, “Missing data imputation and haplotype phase inference forgenome-wide association studies”, Hum Genet (2008) 124:439-450 andBrowning et al. “A unified approach to genotype imputation andhaplotype-phase inference for large data sets of trios and unrelatedindividuals”, Am J Hum Genet. (2009) February; 84(2):210-23, each ofwhich is incorporated herein by reference in its entirety.

The contribution or association of particular SNPs with statin responseor disease phenotypes, such as CVD, enables the SNPs of the presentinvention to be used to develop superior diagnostic tests capable ofidentifying individuals who express a detectable trait, such as reducedrisk for CVD (particularly CHD, such as MI) in response to statintreatment, as the result of a specific genotype, or individuals whosegenotype places them at an increased or decreased risk of developing adetectable trait at a subsequent time as compared to individuals who donot have that genotype. As described herein, diagnostics may be based ona single SNP or a group of SNPs. Combined detection of a plurality ofSNPs (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 24, 25, 30, 32, 48, 50, 64, 96, 100, or any other numberin-between, or more, of the SNPs provided in Table 1 and/or Table 2)typically increases the probability of an accurate diagnosis. Forexample, the presence of a single SNP known to correlate with CVD mightindicate a probability of 20% that an individual has or is at risk ofdeveloping CVD, whereas detection of five SNPs, each of which correlateswith CVD, might indicate a probability of 80% that an individual has oris at risk of developing CVD. To further increase the accuracy ofdiagnosis or predisposition screening, analysis of the SNPs of thepresent invention can be combined with that of other polymorphisms orother risk factors of CVD, such as disease symptoms, pathologicalcharacteristics, family history, diet, environmental factors, orlifestyle factors.

It will be understood by practitioners skilled in the treatment ordiagnosis of CVD that the present invention generally does not intend toprovide an absolute identification of individuals who benefit fromstatin treatment or individuals who are at risk (or less at risk) ofdeveloping CVD, but rather to indicate a certain increased (ordecreased) degree or likelihood of responding to statin therapy ordeveloping CVD based on statistically significant association results.However, this information is extremely valuable as it can be used to,for example, encourage individuals to comply with their statin regimensas prescribed by their doctors (even though the benefit of maintainingstatin therapy may not be overtly apparent, which often leads to lack ofcompliance with prescribed statin treatment), to initiate preventivetreatments or to allow an individual carrying one or more significantSNPs or SNP haplotypes to foresee warning signs such as minor clinicalsymptoms, or to have regularly scheduled physical exams to monitor forappearance of a condition in order to identify and begin treatment ofthe condition at an early stage. Particularly with diseases that areextremely debilitating or fatal if not treated on time, the knowledge ofa potential predisposition, even if this predisposition is not absolute,would likely contribute in a very significant manner to treatmentefficacy.

The diagnostic techniques of the present invention may employ a varietyof methodologies to determine whether a test subject has a SNP orcombination of SNPs associated with an increased or decreased risk ofdeveloping a detectable trait or whether the individual suffers from adetectable trait as a result of a particular polymorphism/mutation,including, for example, methods which enable the analysis of individualchromosomes for haplotyping, family studies, single sperm DNA analysis,or somatic hybrids. The trait analyzed using the diagnostics of theinvention may be any detectable trait that is commonly observed inpathologies and disorders related to CVD or drug response.

Another aspect of the present invention relates to a method ofdetermining whether an individual is at risk (or less at risk) ofdeveloping one or more traits or whether an individual expresses one ormore traits as a consequence of possessing a particular trait-causing ortrait-influencing allele. These methods generally involve obtaining anucleic acid sample from an individual and assaying the nucleic acidsample to determine which nucleotide(s) is/are present at one or moreSNP positions, wherein the assayed nucleotide(s) is/are indicative of anincreased or decreased risk of developing the trait or indicative thatthe individual expresses the trait as a result of possessing aparticular trait-causing or trait-influencing allele.

In another embodiment, the SNP detection reagents of the presentinvention are used to determine whether an individual has one or moreSNP allele(s) affecting the level (e.g., the concentration of mRNA orprotein in a sample, etc.) or pattern (e.g., the kinetics of expression,rate of decomposition, stability profile, Km, Vmax, etc.) of geneexpression (collectively, the “gene response” of a cell or bodilyfluid). Such a determination can be accomplished by screening for mRNAor protein expression (e.g., by using nucleic acid arrays, RT-PCR,TaqMan assays, or mass spectrometry), identifying genes having alteredexpression in an individual, genotyping SNPs disclosed in Table 1 and/orTable 2 that could affect the expression of the genes having alteredexpression (e.g., SNPs that are in and/or around the gene(s) havingaltered expression, SNPs in regulatory/control regions, SNPs in and/oraround other genes that are involved in pathways that could affect theexpression of the gene(s) having altered expression, or all SNPs couldbe genotyped), and correlating SNP genotypes with altered geneexpression. In this manner, specific SNP alleles at particular SNP sitescan be identified that affect gene expression.

Therapeutics, Pharmacogenomics, and Drug Development

Therapeutic Methods and Compositions

In certain aspects of the invention, there are provided methods ofassaying (i.e., testing) one or more SNPs provided by the presentinvention in an individual's nucleic acids, and administering atherapeutic or preventive agent to the individual based on the allele(s)present at the SNP(s) having indicated that the individual can benefitfrom the therapeutic or preventive agent.

In further aspects of the invention, there are provided methods ofassaying one or more SNPs provided by the present invention in anindividual's nucleic acids, and administering a diagnostic agent (e.g.,an imaging agent), or otherwise carrying out further diagnosticprocedures on the individual, based on the allele(s) present at theSNP(s) having indicated that the diagnostic agents or diagnosticsprocedures are justified in the individual.

In yet other aspects of the invention, there is provided apharmaceutical pack comprising a therapeutic agent (e.g., a smallmolecule drug, antibody, peptide, antisense or RNAi nucleic acidmolecule, etc.) and a set of instructions for administration of thetherapeutic agent to an individual who has been tested for one or moreSNPs provided by the present invention.

Pharmacogenomics

The present invention provides methods for assessing thepharmacogenomics of a subject harboring particular SNP alleles orhaplotypes to a particular therapeutic agent or pharmaceutical compound,or to a class of such compounds. Pharmacogenomics deals with the roleswhich clinically significant hereditary variations (e.g., SNPs) play inthe response to drugs due to altered drug disposition and/or abnormalaction in affected persons. See, e.g., Roses, Nature 405, 857-865(2000); Gould Rothberg, Nature Biotechnology 19, 209-211 (2001);Eichelbaum, Clin Exp Pharmacol Physiol 23(10-11):983-985 (1996); andLinder, Clin Chem 43(2):254-266 (1997). The clinical outcomes of thesevariations can result in severe toxicity of therapeutic drugs in certainindividuals or therapeutic failure of drugs in certain individuals as aresult of individual variation in metabolism. Thus, the SNP genotype ofan individual can determine the way a therapeutic compound acts on thebody or the way the body metabolizes the compound. For example, SNPs indrug metabolizing enzymes can affect the activity of these enzymes,which in turn can affect both the intensity and duration of drug action,as well as drug metabolism and clearance.

The discovery of SNPs in drug metabolizing enzymes, drug transporters,proteins for pharmaceutical agents, and other drug targets has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. SNPs can be expressed in the phenotype of the extensivemetabolizer and in the phenotype of the poor metabolizer. Accordingly,SNPs may lead to allelic variants of a protein in which one or more ofthe protein functions in one population are different from those inanother population. SNPs and the encoded variant peptides thus providetargets to ascertain a genetic predisposition that can affect treatmentmodality. For example, in a ligand-based treatment, SNPs may give riseto amino terminal extracellular domains and/or other ligand-bindingregions of a receptor that are more or less active in ligand binding,thereby affecting subsequent protein activation. Accordingly, liganddosage would necessarily be modified to maximize the therapeutic effectwithin a given population containing particular SNP alleles orhaplotypes.

As an alternative to genotyping, specific variant proteins containingvariant amino acid sequences encoded by alternative SNP alleles could beidentified. Thus, pharmacogenomic characterization of an individualpermits the selection of effective compounds and effective dosages ofsuch compounds for prophylactic or therapeutic uses based on theindividual's SNP genotype, thereby enhancing and optimizing theeffectiveness of the therapy. Furthermore, the production of recombinantcells and transgenic animals containing particular SNPs/haplotypes alloweffective clinical design and testing of treatment compounds and dosageregimens. For example, transgenic animals can be produced that differonly in specific SNP alleles in a gene that is orthologous to a humandisease susceptibility gene.

Pharmacogenomic uses of the SNPs of the present invention provideseveral significant advantages for patient care, particularly inpredicting an individual's responsiveness to statin treatment(particularly for reducing the risk of CVD, especially CHD such as MI)and in predicting an individual's predisposition to CVD (e.g., CHD suchas MI). Pharmacogenomic characterization of an individual, based on anindividual's SNP genotype, can identify those individuals unlikely torespond to treatment with a particular medication and thereby allowsphysicians to avoid prescribing the ineffective medication to thoseindividuals. On the other hand, SNP genotyping of an individual mayenable physicians to select the appropriate medication and dosageregimen that will be most effective based on an individual's SNPgenotype. This information increases a physician's confidence inprescribing medications and motivates patients to comply with their drugregimens. Furthermore, pharmacogenomics may identify patientspredisposed to toxicity and adverse reactions to particular drugs ordrug dosages. Adverse drug reactions lead to more than 100,000 avoidabledeaths per year in the United States alone and therefore represent asignificant cause of hospitalization and death, as well as a significanteconomic burden on the healthcare system (Pfost et al., Trends inBiotechnology, August 2000.). Thus, pharmacogenomics based on the SNPsdisclosed herein has the potential to both save lives and reducehealthcare costs substantially.

Pharmacogenomics in general is discussed further in Rose et al.,“Pharmacogenetic analysis of clinically relevant genetic polymorphisms,”Methods Mol Med 85:225-37 (2003). Pharmacogenomics as it relates toAlzheimer's disease and other neurodegenerative disorders is discussedin Cacabelos, “Pharmacogenomics for the treatment of dementia,” Ann Med34(5):357-79 (2002); Maimone et al., “Pharmacogenomics ofneurodegenerative diseases,” Eur J Pharmacol 413(1):11-29 (February2001); and Poirier, “Apolipoprotein E: a pharmacogenetic target for thetreatment of Alzheimer's disease,” Mol Diagn 4(4):335-41 (December1999). Pharmacogenomics as it relates to cardiovascular disorders isdiscussed in Siest et al., “Pharmacogenomics of drugs affecting thecardiovascular system,” Clin Chem Lab Med 41(4):590-9 (April 2003);Mukherjee et al., “Pharmacogenomics in cardiovascular diseases,” ProgCardiovasc Dis 44(6):479-98 (May-June 2002); and Mooser et al.,“Cardiovascular pharmacogenetics in the SNP era,” J Thromb Haemost1(7):1398-402 (July 2003). Pharmacogenomics as it relates to cancer isdiscussed in McLeod et al., “Cancer pharmacogenomics: SNPs, chips, andthe individual patient,” Cancer Invest 21(4):630-40 (2003); and Watterset al., “Cancer pharmacogenomics: current and future applications,”Biochim Biophys Acta 1603(2):99-111 (March 2003).

Clinical Trials

In certain aspects of the invention, there are provided methods of usingthe SNPs disclosed herein to identify or stratify patient populationsfor clinical trials of a therapeutic, preventive, or diagnostic agent.

For instance, an aspect of the present invention includes selectingindividuals for clinical trials based on their SNP genotype, such asselecting individuals for inclusion in a clinical trial and/or assigningindividuals to a particular group within a clinical trial (e.g., an“arm” of the trial). For example, individuals with SNP genotypes thatindicate that they are likely to positively respond to a drug can beincluded in the trials, whereas those individuals whose SNP genotypesindicate that they are less likely to or would not respond to the drug,or who are at risk for suffering toxic effects or other adversereactions, can be excluded from the clinical trials. This not only canimprove the safety of clinical trials, but also can enhance the chancesthat the trial will demonstrate statistically significant efficacy.Further, one can stratify a prospective trial with patients withdifferent SNP variants to determine the impact of differential drugtreatment.

Thus, certain embodiments of the invention provide methods forconducting a clinical trial of a therapeutic agent in which a human isselected for inclusion in the clinical trial and/or assigned to aparticular group within a clinical trial based on the presence orabsence of one or more SNPs disclosed herein. In certain embodiments,the therapeutic agent is a statin.

In certain exemplary embodiments, SNPs of the invention can be used toselect individuals who are unlikely to respond positively to aparticular therapeutic agent (or class of therapeutic agents) based ontheir SNP genotype(s) to participate in a clinical trial of another typeof drug that may benefit them. Thus, in certain embodiments, the SNPs ofthe invention can be used to identify patient populations who do notadequately respond to current treatments and are therefore in need ofnew therapies. This not only benefits the patients themselves, but alsobenefits organizations such as pharmaceutical companies by enabling theidentification of populations that represent markets for new drugs, andenables the efficacy of these new drugs to be tested during clinicaltrials directly in individuals within these markets.

The SNP-containing nucleic acid molecules of the present invention arealso useful for monitoring the effectiveness of modulating compounds onthe expression or activity of a variant gene, or encoded product,particularly in a treatment regimen or in clinical trials. Thus, thegene expression pattern can serve as an indicator for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance, as well as anindicator for toxicities. The gene expression pattern can also serve asa marker indicative of a physiological response of the affected cells tothe compound. Accordingly, such monitoring would allow either increasedadministration of the compound or the administration of alternativecompounds to which the patient has not become resistant.

Furthermore, the SNPs of the present invention may have utility indetermining why certain previously developed drugs performed poorly inclinical trials and may help identify a subset of the population thatwould benefit from a drug that had previously performed poorly inclinical trials, thereby “rescuing” previously developed drugs, andenabling the drug to be made available to a particular patientpopulation (e.g., particular CVD patients) that can benefit from it.

Identification, Screening, and Use of Therapeutic Agents

The SNPs of the present invention also can be used to identify noveltherapeutic targets for CVD, particularly CHD, such as MI, or stroke.For example, genes containing the disease-associated variants (“variantgenes”) or their products, as well as genes or their products that aredirectly or indirectly regulated by or interacting with these variantgenes or their products, can be targeted for the development oftherapeutics that, for example, treat the disease or prevent or delaydisease onset. The therapeutics may be composed of, for example, smallmolecules, proteins, protein fragments or peptides, antibodies, nucleicacids, or their derivatives or mimetics which modulate the functions orlevels of the target genes or gene products.

The invention further provides methods for identifying a compound oragent that can be used to treat CVD, particularly CHD such as MI. TheSNPs disclosed herein are useful as targets for the identificationand/or development of therapeutic agents. A method for identifying atherapeutic agent or compound typically includes assaying the ability ofthe agent or compound to modulate the activity and/or expression of aSNP-containing nucleic acid or the encoded product and thus identifyingan agent or a compound that can be used to treat a disordercharacterized by undesired activity or expression of the SNP-containingnucleic acid or the encoded product. The assays can be performed incell-based and cell-free systems. Cell-based assays can include cellsnaturally expressing the nucleic acid molecules of interest orrecombinant cells genetically engineered to express certain nucleic acidmolecules.

Variant gene expression in a CVD patient can include, for example,either expression of a SNP-containing nucleic acid sequence (forinstance, a gene that contains a SNP can be transcribed into an mRNAtranscript molecule containing the SNP, which can in turn be translatedinto a variant protein) or altered expression of a normal/wild-typenucleic acid sequence due to one or more SNPs (for instance, aregulatory/control region can contain a SNP that affects the level orpattern of expression of a normal transcript).

Assays for variant gene expression can involve direct assays of nucleicacid levels (e.g., mRNA levels), expressed protein levels, or ofcollateral compounds involved in a signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thesignal pathway can also be assayed. In this embodiment, the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

Modulators of variant gene expression can be identified in a methodwherein, for example, a cell is contacted with a candidatecompound/agent and the expression of mRNA determined. The level ofexpression of mRNA in the presence of the candidate compound is comparedto the level of expression of mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof variant gene expression based on this comparison and be used to treata disorder such as CVD that is characterized by variant gene expression(e.g., either expression of a SNP-containing nucleic acid or alteredexpression of a normal/wild-type nucleic acid molecule due to one ormore SNPs that affect expression of the nucleic acid molecule) due toone or more SNPs of the present invention. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the SNP orassociated nucleic acid domain (e.g., catalytic domain,ligand/substrate-binding domain, regulatory/control region, etc.) orgene, or the encoded mRNA transcript, as a target, using a compoundidentified through drug screening as a gene modulator to modulatevariant nucleic acid expression. Modulation can include eitherup-regulation (i.e., activation or agonization) or down-regulation(i.e., suppression or antagonization) of nucleic acid expression.

Expression of mRNA transcripts and encoded proteins, either wild type orvariant, may be altered in individuals with a particular SNP allele in aregulatory/control element, such as a promoter or transcription factorbinding domain, that regulates expression. In this situation, methods oftreatment and compounds can be identified, as discussed herein, thatregulate or overcome the variant regulatory/control element, therebygenerating normal, or healthy, expression levels of either the wild typeor variant protein.

Pharmaceutical Compositions and Administration Thereof

Any of the statin response-associated proteins, and encoding nucleicacid molecules, disclosed herein can be used as therapeutic targets (ordirectly used themselves as therapeutic compounds) for treating orpreventing CVD, and the present disclosure enables therapeutic compounds(e.g., small molecules, antibodies, therapeutic proteins, RNAi andantisense molecules, etc.) to be developed that target (or are comprisedof) any of these therapeutic targets.

In general, a therapeutic compound will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the therapeutic compound of this invention, i.e., the activeingredient, will depend upon numerous factors such as the severity ofthe disease to be treated, the age and relative health of the subject,the potency of the compound used, the route and form of administration,and other factors.

Therapeutically effective amounts of therapeutic compounds may rangefrom, for example, approximately 0.01-50 mg per kilogram body weight ofthe recipient per day; preferably about 0.1-20 mg/kg/day. Thus, as anexample, for administration to a 70-kg person, the dosage range wouldmost preferably be about 7 mg to 1.4 g per day.

In general, therapeutic compounds will be administered as pharmaceuticalcompositions by any one of the following routes: oral, systemic (e.g.,transdermal, intranasal, or by suppository), or parenteral (e.g.,intramuscular, intravenous, or subcutaneous) administration. Thepreferred manner of administration is oral or parenteral using aconvenient daily dosage regimen, which can be adjusted according to thedegree of affliction. Oral compositions can take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions.

The choice of formulation depends on various factors such as the mode ofdrug administration (e.g., for oral administration, formulations in theform of tablets, pills, or capsules are preferred) and thebioavailability of the drug substance. Recently, pharmaceuticalformulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area, i.e., decreasing particlesize. For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalformulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a cross-linked matrix ofmacromolecules. U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

Pharmaceutical compositions are comprised of, in general, a therapeuticcompound in combination with at least one pharmaceutically acceptableexcipient. Acceptable excipients are non-toxic, aid administration, anddo not adversely affect the therapeutic benefit of the therapeuticcompound. Such excipients may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one skilled in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc.

Other suitable pharmaceutical excipients and their formulations aredescribed in Remington's Pharmaceutical Sciences 18^(th) ed., E. W.Martin, ed., Mack Publishing Company (1990).

The amount of the therapeutic compound in a formulation can vary withinthe full range employed by those skilled in the art. Typically, theformulation will contain, on a weight percent (wt %) basis, from about0.01-99.99 wt % of the therapeutic compound based on the totalformulation, with the balance being one or more suitable pharmaceuticalexcipients. Preferably, the compound is present at a level of about1-80% wt.

Therapeutic compounds can be administered alone or in combination withother therapeutic compounds or in combination with one or more otheractive ingredient(s). For example, an inhibitor or stimulator of aCVD-associated protein can be administered in combination with anotheragent that inhibits or stimulates the activity of the same or adifferent CVD-associated protein to thereby counteract the effects ofCVD.

For further information regarding pharmacology, see Current Protocols inPharmacology, John Wiley & Sons, Inc., N.Y.

Nucleic Acid-Based Therapeutic Agents

The SNP-containing nucleic acid molecules disclosed herein, and theircomplementary nucleic acid molecules, may be used as antisenseconstructs to control gene expression in cells, tissues, and organisms.Antisense technology is well established in the art and extensivelyreviewed in Antisense Drug Technology: Principles, Strategies, andApplications, Crooke, ed., Marcel Dekker, Inc., N.Y. (2001). Anantisense nucleic acid molecule is generally designed to becomplementary to a region of mRNA expressed by a gene so that theantisense molecule hybridizes to the mRNA and thereby blocks translationof mRNA into protein. Various classes of antisense oligonucleotides areused in the art, two of which are cleavers and blockers. Cleavers, bybinding to target RNAs, activate intracellular nucleases (e.g., RNaseHor RNase L) that cleave the target RNA. Blockers, which also bind totarget RNAs, inhibit protein translation through steric hindrance ofribosomes. Exemplary blockers include peptide nucleic acids,morpholinos, locked nucleic acids, and methylphosphonates. See, e.g.,Thompson, Drug Discovery Today 7(17): 912-917 (2002). Antisenseoligonucleotides are directly useful as therapeutic agents, and are alsouseful for determining and validating gene function (e.g., in geneknock-out or knock-down experiments).

Antisense technology is further reviewed in: Lavery et al., “Antisenseand RNAi: powerful tools in drug target discovery and validation,” CurrOpin Drug Discov Devel 6(4):561-9 (July 2003); Stephens et al.,“Antisense oligonucleotide therapy in cancer,” Curr Opin Mol Ther5(2):118-22 (April 2003); Kurreck, “Antisense technologies. Improvementthrough novel chemical modifications,” Eur J Biochem 270(8):1628-44(April 2003); Dias et al., “Antisense oligonucleotides: basic conceptsand mechanisms,” Mol Cancer Ther 1(5):347-55 (March 2002); Chen,“Clinical development of antisense oligonucleotides as anti-cancertherapeutics,” Methods Mol Med 75:621-36 (2003); Wang et al., “Antisenseanticancer oligonucleotide therapeutics,” Curr Cancer Drug Targets1(3):177-96 (November 2001); and Bennett, “Efficiency of antisenseoligonucleotide drug discovery,” Antisense Nucleic Acid Drug Dev12(3):215-24 (June 2002).

The SNPs of the present invention are particularly useful for designingantisense reagents that are specific for particular nucleic acidvariants. Based on the SNP information disclosed herein, antisenseoligonucleotides can be produced that specifically target mRNA moleculesthat contain one or more particular SNP nucleotides. In this manner,expression of mRNA molecules that contain one or more undesiredpolymorphisms (e.g., SNP nucleotides that lead to a defective proteinsuch as an amino acid substitution in a catalytic domain) can beinhibited or completely blocked. Thus, antisense oligonucleotides can beused to specifically bind a particular polymorphic form (e.g., a SNPallele that encodes a defective protein), thereby inhibiting translationof this form, but which do not bind an alternative polymorphic form(e.g., an alternative SNP nucleotide that encodes a protein havingnormal function).

Antisense molecules can be used to inactivate mRNA in order to inhibitgene expression and production of defective proteins. Accordingly, thesemolecules can be used to treat a disorder, such as CVD, characterized byabnormal or undesired gene expression or expression of certain defectiveproteins. This technique can involve cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible mRNA regions include, for example, protein-coding regions andparticularly protein-coding regions corresponding to catalyticactivities, substrate/ligand binding, or other functional activities ofa protein.

The SNPs of the present invention are also useful for designing RNAinterference reagents that specifically target nucleic acid moleculeshaving particular SNP variants. RNA interference (RNAi), also referredto as gene silencing, is based on using double-stranded RNA (dsRNA)molecules to turn genes off. When introduced into a cell, dsRNAs areprocessed by the cell into short fragments (generally about 21, 22, or23 nucleotides in length) known as small interfering RNAs (siRNAs) whichthe cell uses in a sequence-specific manner to recognize and destroycomplementary RNAs. Thompson, Drug Discovery Today 7(17): 912-917(2002). Accordingly, an aspect of the present invention specificallycontemplates isolated nucleic acid molecules that are about 18-26nucleotides in length, preferably 19-25 nucleotides in length, and morepreferably 20, 21, 22, or 23 nucleotides in length, and the use of thesenucleic acid molecules for RNAi. Because RNAi molecules, includingsiRNAs, act in a sequence-specific manner, the SNPs of the presentinvention can be used to design RNAi reagents that recognize and destroynucleic acid molecules having specific SNP alleles/nucleotides (such asdeleterious alleles that lead to the production of defective proteins),while not affecting nucleic acid molecules having alternative SNPalleles (such as alleles that encode proteins having normal function).As with antisense reagents, RNAi reagents may be directly useful astherapeutic agents (e.g., for turning off defective, disease-causinggenes), and are also useful for characterizing and validating genefunction (e.g., in gene knock-out or knock-down experiments).

The following references provide a further review of RNAi: Reynolds etal., “Rational siRNA design for RNA interference,” Nat Biotechnol22(3):326-30 (March 2004); Epub Feb. 1, 2004; Chi et al., “Genomewideview of gene silencing by small interfering RNAs,” PNAS100(11):6343-6346 (2003); Vickers et al., “Efficient Reduction of TargetRNAs by Small Interfering RNA and RNase H-dependent Antisense Agents,” JBiol Chem 278:7108-7118 (2003); Agami, “RNAi and related mechanisms andtheir potential use for therapy,” Curr Opin Chem Biol 6(6):829-34(December 2002); Lavery et al., “Antisense and RNAi: powerful tools indrug target discovery and validation,” Curr Opin Drug Discov Devel6(4):561-9 (July 2003); Shi, “Mammalian RNAi for the masses,” TrendsGenet 19(1):9-12 (January 2003); Shuey et al., “RNAi: gene-silencing intherapeutic intervention,” Drug Discovery Today 7(20):1040-1046 (October2002); McManus et al., Nat Rev Genet 3(10):737-47 (October 2002); Xia etal., Nat Biotechnol 20(10):1006-10 (October 2002); Plasterk et al., CurrOpin Genet Dev 10(5):562-7 (October 2000); Bosher et al., Nat Cell Biol2(2):E31-6 (February 2000); and Hunter, Curr Biol 17; 9(12):R440-2 (June1999).

Other Therapeutic Aspects

SNPs have many important uses in drug discovery, screening, anddevelopment, and thus the SNPs of the present invention are useful forimproving many different aspects of the drug development process.

For example, a high probability exists that, for any gene/proteinselected as a potential drug target, variants of that gene/protein willexist in a patient population. Thus, determining the impact ofgene/protein variants on the selection and delivery of a therapeuticagent should be an integral aspect of the drug discovery and developmentprocess. Jazwinska, A Trends Guide to Genetic Variation and GenomicMedicine S30-S36 (March 2002).

Knowledge of variants (e.g., SNPs and any corresponding amino acidpolymorphisms) of a particular therapeutic target (e.g., a gene, mRNAtranscript, or protein) enables parallel screening of the variants inorder to identify therapeutic candidates (e.g., small moleculecompounds, antibodies, antisense or RNAi nucleic acid compounds, etc.)that demonstrate efficacy across variants. Rothberg, Nat Biotechnol19(3):209-11 (March 2001). Such therapeutic candidates would be expectedto show equal efficacy across a larger segment of the patientpopulation, thereby leading to a larger potential market for thetherapeutic candidate.

Furthermore, identifying variants of a potential therapeutic targetenables the most common form of the target to be used for selection oftherapeutic candidates, thereby helping to ensure that the experimentalactivity that is observed for the selected candidates reflects the realactivity expected in the largest proportion of a patient population.Jazwinska, A Trends Guide to Genetic Variation and Genomic MedicineS30-S36 (March 2002).

Additionally, screening therapeutic candidates against all knownvariants of a target can enable the early identification of potentialtoxicities and adverse reactions relating to particular variants. Forexample, variability in drug absorption, distribution, metabolism andexcretion (ADME) caused by, for example, SNPs in therapeutic targets ordrug metabolizing genes, can be identified, and this information can beutilized during the drug development process to minimize variability indrug disposition and develop therapeutic agents that are safer across awider range of a patient population. The SNPs of the present invention,including the variant proteins and encoding polymorphic nucleic acidmolecules provided in Tables 1 and 2, are useful in conjunction with avariety of toxicology methods established in the art, such as those setforth in Current Protocols in Toxicology, John Wiley & Sons, Inc., N.Y.

Furthermore, therapeutic agents that target any art-known proteins (ornucleic acid molecules, either RNA or DNA) may cross-react with thevariant proteins (or polymorphic nucleic acid molecules) disclosed inTable 1, thereby significantly affecting the pharmacokinetic propertiesof the drug. Consequently, the protein variants and the SNP-containingnucleic acid molecules disclosed in Tables 1 and 2 are useful indeveloping, screening, and evaluating therapeutic agents that targetcorresponding art-known protein forms (or nucleic acid molecules).Additionally, as discussed above, knowledge of all polymorphic forms ofa particular drug target enables the design of therapeutic agents thatare effective against most or all such polymorphic forms of the drugtarget.

A subject suffering from a pathological condition ascribed to a SNP,such as CVD, may be treated so as to correct the genetic defect. SeeKren et al., Proc Natl Acad Sci USA 96:10349-10354 (1999). Such asubject can be identified by any method that can detect the polymorphismin a biological sample drawn from the subject. Such a genetic defect maybe permanently corrected by administering to such a subject a nucleicacid fragment incorporating a repair sequence that supplies thenormal/wild-type nucleotide at the position of the SNP. Thissite-specific repair sequence can encompass an RNA/DNA oligonucleotidethat operates to promote endogenous repair of a subject's genomic DNA.The site-specific repair sequence is administered in an appropriatevehicle, such as a complex with polyethylenimine, encapsulated inanionic liposomes, a viral vector such as an adenovirus, or otherpharmaceutical composition that promotes intracellular uptake of theadministered nucleic acid. A genetic defect leading to an inbornpathology may then be overcome, as the chimeric oligonucleotides induceincorporation of the normal sequence into the subject's genome. Uponincorporation, the normal gene product is expressed, and the replacementis propagated, thereby engendering a permanent repair and therapeuticenhancement of the clinical condition of the subject.

In cases in which a cSNP results in a variant protein that is ascribedto be the cause of, or a contributing factor to, a pathologicalcondition, a method of treating such a condition can includeadministering to a subject experiencing the pathology thewild-type/normal cognate of the variant protein. Once administered in aneffective dosing regimen, the wild-type cognate provides complementationor remediation of the pathological condition.

Variant Proteins, Antibodies, Vectors, Host Cells, & Uses Thereof

Variant Proteins Encoded by SNP-Containing Nucleic Acid Molecules

The present invention provides SNP-containing nucleic acid molecules,many of which encode proteins having variant amino acid sequences ascompared to the art-known (i.e., wild-type) proteins. Amino acidsequences encoded by the polymorphic nucleic acid molecules of thepresent invention are referred to as SEQ ID NOS:52-102 in Table 1 andprovided in the Sequence Listing. These variants will generally bereferred to herein as variant proteins/peptides/polypeptides, orpolymorphic proteins/peptides/polypeptides of the present invention. Theterms “protein,” “peptide,” and “polypeptide” are used hereininterchangeably.

A variant protein of the present invention may be encoded by, forexample, a nonsynonymous nucleotide substitution at any one of the cSNPpositions disclosed herein. In addition, variant proteins may alsoinclude proteins whose expression, structure, and/or function is alteredby a SNP disclosed herein, such as a SNP that creates or destroys a stopcodon, a SNP that affects splicing, and a SNP in control/regulatoryelements, e.g. promoters, enhancers, or transcription factor bindingdomains.

As used herein, a protein or peptide is said to be “isolated” or“purified” when it is substantially free of cellular material orchemical precursors or other chemicals. The variant proteins of thepresent invention can be purified to homogeneity or other lower degreesof purity. The level of purification will be based on the intended use.The key feature is that the preparation allows for the desired functionof the variant protein, even if in the presence of considerable amountsof other components.

As used herein, “substantially free of cellular material” includespreparations of the variant protein having less than about 30% (by dryweight) other proteins (i.e., contaminating protein), less than about20% other proteins, less than about 10% other proteins, or less thanabout 5% other proteins. When the variant protein is recombinantlyproduced, it can also be substantially free of culture medium, i.e.,culture medium represents less than about 20% of the volume of theprotein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the variant protein in which it isseparated from chemical precursors or other chemicals that are involvedin its synthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thevariant protein having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

An isolated variant protein may be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant host cells), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule containing SNP(s) encodingthe variant protein can be cloned into an expression vector, theexpression vector introduced into a host cell, and the variant proteinexpressed in the host cell. The variant protein can then be isolatedfrom the cells by any appropriate purification scheme using standardprotein purification techniques. Examples of these techniques aredescribed in detail below. Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (2000).

The present invention provides isolated variant proteins that comprise,consist of or consist essentially of amino acid sequences that containone or more variant amino acids encoded by one or more codons thatcontain a SNP of the present invention.

Accordingly, the present invention provides variant proteins thatconsist of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists of an amino acid sequencewhen the amino acid sequence is the entire amino acid sequence of theprotein.

The present invention further provides variant proteins that consistessentially of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists essentially of an aminoacid sequence when such an amino acid sequence is present with only afew additional amino acid residues in the final protein.

The present invention further provides variant proteins that compriseamino acid sequences that contain one or more amino acid polymorphisms(or truncations or extensions due to creation or destruction of a stopcodon, respectively) encoded by the SNPs provided in Table 1 and/orTable 2. A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein may contain only the variantamino acid sequence or have additional amino acid residues, such as acontiguous encoded sequence that is naturally associated with it orheterologous amino acid residues. Such a protein can have a fewadditional amino acid residues or can comprise many more additionalamino acids. A brief description of how various types of these proteinscan be made and isolated is provided below.

The variant proteins of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a variant protein operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the variant protein. “Operatively linked”indicates that the coding sequences for the variant protein and theheterologous protein are ligated in-frame. The heterologous protein canbe fused to the N-terminus or C-terminus of the variant protein. Inanother embodiment, the fusion protein is encoded by a fusionpolynucleotide that is synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and re-amplified to generate a chimeric genesequence. See Ausubel et al., Current Protocols in Molecular Biology(1992). Moreover, many expression vectors are commercially availablethat already encode a fusion moiety (e.g., a GST protein). A variantprotein-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the variantprotein.

In many uses, the fusion protein does not affect the activity of thevariant protein. The fusion protein can include, but is not limited to,enzymatic fusion proteins, for example, beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-taggedand Ig fusions. Such fusion proteins, particularly poly-His fusions, canfacilitate their purification following recombinant expression. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence. Fusion proteins are further described in, for example, Terpe,“Overview of tag protein fusions: from molecular and biochemicalfundamentals to commercial systems,” Appl Microbiol Biotechnol60(5):523-33 (January 2003); Epub Nov. 7, 2002; Graddis et al.,“Designing proteins that work using recombinant technologies,” CurrPharm Biotechnol 3(4):285-97 (December 2002); and Nilsson et al.,“Affinity fusion strategies for detection, purification, andimmobilization of recombinant proteins,” Protein Expr Purif 11(1):1-16(October 1997).

In certain embodiments, novel compositions of the present invention alsorelate to further obvious variants of the variant polypeptides of thepresent invention, such as naturally-occurring mature forms (e.g.,allelic variants), non-naturally occurring recombinantly-derivedvariants, and orthologs and paralogs of such proteins that sharesequence homology. Such variants can readily be generated usingart-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry.

Further variants of the variant polypeptides disclosed in Table 1 cancomprise an amino acid sequence that shares at least 70-80%, 80-85%,85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitywith an amino acid sequence disclosed in Table 1 (or a fragment thereof)and that includes a novel amino acid residue (allele) disclosed in Table1 (which is encoded by a novel SNP allele). Thus, an aspect of thepresent invention that is specifically contemplated are polypeptidesthat have a certain degree of sequence variation compared with thepolypeptide sequences shown in Table 1, but that contain a novel aminoacid residue (allele) encoded by a novel SNP allele disclosed herein. Inother words, as long as a polypeptide contains a novel amino acidresidue disclosed herein, other portions of the polypeptide that flankthe novel amino acid residue can vary to some degree from thepolypeptide sequences shown in Table 1.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the amino acid sequences disclosed hereincan readily be identified as having complete sequence identity to one ofthe variant proteins of the present invention as well as being encodedby the same genetic locus as the variant proteins provided herein.

Orthologs of a variant peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof a variant peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from non-human mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs can be encoded by a nucleic acid sequencethat hybridizes to a variant peptide-encoding nucleic acid moleculeunder moderate to stringent conditions depending on the degree ofrelatedness of the two organisms yielding the homologous proteins.

Variant proteins include, but are not limited to, proteins containingdeletions, additions and substitutions in the amino acid sequence causedby the SNPs of the present invention. One class of substitutions isconserved amino acid substitutions in which a given amino acid in apolypeptide is substituted for another amino acid of likecharacteristics. Typical conservative substitutions are replacements,one for another, among the aliphatic amino acids Ala, Val, Leu, and Be;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found, for example,in Bowie et al., Science 247:1306-1310 (1990).

Variant proteins can be fully functional or can lack function in one ormore activities, e.g. ability to bind another molecule, ability tocatalyze a substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, truncations or extensions, or a substitution,insertion, inversion, or deletion of a critical residue or in a criticalregion.

Amino acids that are essential for function of a protein can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis, particularly using theamino acid sequence and polymorphism information provided in Table 1.Cunningham et al., Science 244:1081-1085 (1989). The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as enzyme activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling. Smith et al., JMol Biol 224:899-904 (1992); de Vos et al., Science 255:306-312 (1992).

Polypeptides can contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Accordingly, the variant proteins of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (e.g., polyethylene glycol), or in which additional aminoacids are fused to the mature polypeptide, such as a leader or secretorysequence or a sequence for purification of the mature polypeptide or apro-protein sequence.

Known protein modifications include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such protein modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Particularly common modifications, for example glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, are described in most basic texts,such as Proteins—Structure and Molecular Properties 2nd Ed., T. E.Creighton, W.H. Freeman and Company, N.Y. (1993); F. Wold,Posttranslational Covalent Modification of Proteins 1-12, B. C. Johnson,ed., Academic Press, N.Y. (1983); Seifter et al., Meth Enzymol182:626-646 (1990); and Rattan et al., Ann NY Acad Sci 663:48-62 (1992).

The present invention further provides fragments of the variant proteinsin which the fragments contain one or more amino acid sequencevariations (e.g., substitutions, or truncations or extensions due tocreation or destruction of a stop codon) encoded by one or more SNPsdisclosed herein. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that havebeen disclosed in the prior art before the present invention.

As used herein, a fragment may comprise at least about 4, 8, 10, 12, 14,16, 18, 20, 25, 30, 50, 100 (or any other number in-between) or morecontiguous amino acid residues from a variant protein, wherein at leastone amino acid residue is affected by a SNP of the present invention,e.g., a variant amino acid residue encoded by a nonsynonymous nucleotidesubstitution at a cSNP position provided by the present invention. Thevariant amino acid encoded by a cSNP may occupy any residue positionalong the sequence of the fragment. Such fragments can be chosen basedon the ability to retain one or more of the biological activities of thevariant protein or the ability to perform a function, e.g., act as animmunogen. Particularly important fragments are biologically activefragments. Such fragments will typically comprise a domain or motif of avariant protein of the present invention, e.g., active site,transmembrane domain, or ligand/substrate binding domain. Otherfragments include, but are not limited to, domain or motif-containingfragments, soluble peptide fragments, and fragments containingimmunogenic structures. Predicted domains and functional sites arereadily identifiable by computer programs well known to those of skillin the art (e.g., PROSITE analysis). Current Protocols in ProteinScience, John Wiley & Sons, N.Y. (2002).

Uses of Variant Proteins

The variant proteins of the present invention can be used in a varietyof ways, including but not limited to, in assays to determine thebiological activity of a variant protein, such as in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother type of immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of thevariant protein (or its binding partner) in biological fluids; as amarker for cells or tissues in which it is preferentially expressed(either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); as a target forscreening for a therapeutic agent; and as a direct therapeutic agent tobe administered into a human subject. Any of the variant proteinsdisclosed herein may be developed into reagent grade or kit format forcommercialization as research products. Methods for performing the useslisted above are well known to those skilled in the art. See, e.g.,Molecular Cloning: A Laboratory Manual, Sambrook and Russell, ColdSpring Harbor Laboratory Press, N.Y. (2000), and Methods in Enzymology:Guide to Molecular Cloning Techniques, S. L. Berger and A. R. Kimmel,eds., Academic Press (1987).

In a specific embodiment of the invention, the methods of the presentinvention include detection of one or more variant proteins disclosedherein. Variant proteins are disclosed in Table 1 and in the SequenceListing as SEQ ID NOS:52-102. Detection of such proteins can beaccomplished using, for example, antibodies, small molecule compounds,aptamers, ligands/substrates, other proteins or protein fragments, orother protein-binding agents. Preferably, protein detection agents arespecific for a variant protein of the present invention and cantherefore discriminate between a variant protein of the presentinvention and the wild-type protein or another variant form. This cangenerally be accomplished by, for example, selecting or designingdetection agents that bind to the region of a protein that differsbetween the variant and wild-type protein, such as a region of a proteinthat contains one or more amino acid substitutions that is/are encodedby a non-synonymous cSNP of the present invention, or a region of aprotein that follows a nonsense mutation-type SNP that creates a stopcodon thereby leading to a shorter polypeptide, or a region of a proteinthat follows a read-through mutation-type SNP that destroys a stop codonthereby leading to a longer polypeptide in which a portion of thepolypeptide is present in one version of the polypeptide but not theother.

In another aspect of the invention, variant proteins of the presentinvention can be used as targets for predicting an individual's responseto statin treatment (particularly for reducing the risk of CVD,especially CHD such as MI), for determining predisposition to CVD(particularly CHD, such as MI), for diagnosing CVD, or for treatingand/or preventing CVD, etc. Accordingly, the invention provides methodsfor detecting the presence of, or levels of, one or more variantproteins of the present invention in a cell, tissue, or organism. Suchmethods typically involve contacting a test sample with an agent (e.g.,an antibody, small molecule compound, or peptide) capable of interactingwith the variant protein such that specific binding of the agent to thevariant protein can be detected. Such an assay can be provided in asingle detection format or a multi-detection format such as an array,for example, an antibody or aptamer array (arrays for protein detectionmay also be referred to as “protein chips”). The variant protein ofinterest can be isolated from a test sample and assayed for the presenceof a variant amino acid sequence encoded by one or more SNPs disclosedby the present invention. The SNPs may cause changes to the protein andthe corresponding protein function/activity, such as throughnon-synonymous substitutions in protein coding regions that can lead toamino acid substitutions, deletions, insertions, and/or rearrangements;formation or destruction of stop codons; or alteration of controlelements such as promoters. SNPs may also cause inappropriatepost-translational modifications.

One preferred agent for detecting a variant protein in a sample is anantibody capable of selectively binding to a variant form of the protein(antibodies are described in greater detail in the next section). Suchsamples include, for example, tissues, cells, and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject.

In vitro methods for detection of the variant proteins associated withstatin response that are disclosed herein and fragments thereof include,but are not limited to, enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), Western blots, immunoprecipitations,immunofluorescence, and protein arrays/chips (e.g., arrays of antibodiesor aptamers). For further information regarding immunoassays and relatedprotein detection methods, see Current Protocols in Immunology, JohnWiley & Sons, N.Y., and Hage, “Immunoassays,” Anal Chem 15;71(12):294R-304R (June 1999).

Additional analytic methods of detecting amino acid variants include,but are not limited to, altered electrophoretic mobility, alteredtryptic peptide digest, altered protein activity in cell-based orcell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, and direct amino acid sequencing.

Alternatively, variant proteins can be detected in vivo in a subject byintroducing into the subject a labeled antibody (or other type ofdetection reagent) specific for a variant protein. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Other uses of the variant peptides of the present invention are based onthe class or action of the protein. For example, proteins isolated fromhumans and their mammalian orthologs serve as targets for identifyingagents (e.g., small molecule drugs or antibodies) for use in therapeuticapplications, particularly for modulating a biological or pathologicalresponse in a cell or tissue that expresses the protein. Pharmaceuticalagents can be developed that modulate protein activity.

As an alternative to modulating gene expression, therapeutic compoundscan be developed that modulate protein function. For example, many SNPsdisclosed herein affect the amino acid sequence of the encoded protein(e.g., non-synonymous cSNPs and nonsense mutation-type SNPs). Suchalterations in the encoded amino acid sequence may affect proteinfunction, particularly if such amino acid sequence variations occur infunctional protein domains, such as catalytic domains, ATP-bindingdomains, or ligand/substrate binding domains. It is well established inthe art that variant proteins having amino acid sequence variations infunctional domains can cause or influence pathological conditions. Insuch instances, compounds (e.g., small molecule drugs or antibodies) canbe developed that target the variant protein and modulate (e.g., up- ordown-regulate) protein function/activity.

The therapeutic methods of the present invention further include methodsthat target one or more variant proteins of the present invention.Variant proteins can be targeted using, for example, small moleculecompounds, antibodies, aptamers, ligands/substrates, other proteins, orother protein-binding agents. Additionally, the skilled artisan willrecognize that the novel protein variants (and polymorphic nucleic acidmolecules) disclosed in Table 1 may themselves be directly used astherapeutic agents by acting as competitive inhibitors of correspondingart-known proteins (or nucleic acid molecules such as mRNA molecules).

The variant proteins of the present invention are particularly useful indrug screening assays, in cell-based or cell-free systems. Cell-basedsystems can utilize cells that naturally express the protein, a biopsyspecimen, or cell cultures. In one embodiment, cell-based assays involverecombinant host cells expressing the variant protein. Cell-free assayscan be used to detect the ability of a compound to directly bind to avariant protein or to the corresponding SNP-containing nucleic acidfragment that encodes the variant protein.

A variant protein of the present invention, as well as appropriatefragments thereof, can be used in high-throughput screening assays totest candidate compounds for the ability to bind and/or modulate theactivity of the variant protein. These candidate compounds can befurther screened against a protein having normal function (e.g., awild-type/non-variant protein) to further determine the effect of thecompound on the protein activity. Furthermore, these compounds can betested in animal or invertebrate systems to determine in vivoactivity/effectiveness. Compounds can be identified that activate(agonists) or inactivate (antagonists) the variant protein, anddifferent compounds can be identified that cause various degrees ofactivation or inactivation of the variant protein.

Further, the variant proteins can be used to screen a compound for theability to stimulate or inhibit interaction between the variant proteinand a target molecule that normally interacts with the protein. Thetarget can be a ligand, a substrate or a binding partner that theprotein normally interacts with (for example, epinephrine ornorepinephrine). Such assays typically include the steps of combiningthe variant protein with a candidate compound under conditions thatallow the variant protein, or fragment thereof, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the variant protein and the target, such as any of theassociated effects of signal transduction.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the variant protein thatcompetes for ligand binding. Other candidate compounds include mutantproteins or appropriate fragments containing mutations that affectvariant protein function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) variant protein activity.The assays typically involve an assay of events in the signaltransduction pathway that indicate protein activity. Thus, theexpression of genes that are up or down-regulated in response to thevariant protein dependent signal cascade can be assayed. In oneembodiment, the regulatory region of such genes can be operably linkedto a marker that is easily detectable, such as luciferase.Alternatively, phosphorylation of the variant protein, or a variantprotein target, could also be measured. Any of the biological orbiochemical functions mediated by the variant protein can be used as anendpoint assay. These include all of the biochemical or biologicalevents described herein, in the references cited herein, incorporated byreference for these endpoint assay targets, and other functions known tothose of ordinary skill in the art.

Binding and/or activating compounds can also be screened by usingchimeric variant proteins in which an amino terminal extracellulardomain or parts thereof, an entire transmembrane domain or subregions,and/or the carboxyl terminal intracellular domain or parts thereof, canbe replaced by heterologous domains or subregions. For example, asubstrate-binding region can be used that interacts with a differentsubstrate than that which is normally recognized by a variant protein.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which thevariant protein is derived.

The variant proteins are also useful in competition binding assays inmethods designed to discover compounds that interact with the variantprotein. Thus, a compound can be exposed to a variant protein underconditions that allow the compound to bind or to otherwise interact withthe variant protein. A binding partner, such as ligand, that normallyinteracts with the variant protein is also added to the mixture. If thetest compound interacts with the variant protein or its binding partner,it decreases the amount of complex formed or activity from the variantprotein. This type of assay is particularly useful in screening forcompounds that interact with specific regions of the variant protein.Hodgson, Bio/technology, 10(9), 973-80 (September 1992).

To perform cell-free drug screening assays, it is sometimes desirable toimmobilize either the variant protein or a fragment thereof, or itstarget molecule, to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Any method for immobilizing proteins onmatrices can be used in drug screening assays. In one embodiment, afusion protein containing an added domain allows the protein to be boundto a matrix. For example, glutathione-S-transferase/¹²⁵I fusion proteinscan be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, MO) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and a candidatecompound, such as a drug candidate, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads can bewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofbound material found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Either the variant protein or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Alternatively,antibodies reactive with the variant protein but which do not interferewith binding of the variant protein to its target molecule can bederivatized to the wells of the plate, and the variant protein trappedin the wells by antibody conjugation. Preparations of the targetmolecule and a candidate compound are incubated in the variantprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the proteintarget molecule, or which are reactive with variant protein and competewith the target molecule, and enzyme-linked assays that rely ondetecting an enzymatic activity associated with the target molecule.

Modulators of variant protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the protein pathway, such as CVD. These methods of treatmenttypically include the steps of administering the modulators of proteinactivity in a pharmaceutical composition to a subject in need of suchtreatment.

The variant proteins, or fragments thereof, disclosed herein canthemselves be directly used to treat a disorder characterized by anabsence of, inappropriate, or unwanted expression or activity of thevariant protein. Accordingly, methods for treatment include the use of avariant protein disclosed herein or fragments thereof.

In yet another aspect of the invention, variant proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay to identifyother proteins that bind to or interact with the variant protein and areinvolved in variant protein activity. See, e.g., U.S. Pat. No.5,283,317; Zervos et al., Cell 72:223-232 (1993); Madura et al., J BiolChem 268:12046-12054 (1993); Bartel et al., Biotechniques 14:920-924(1993); Iwabuchi et al., Oncogene 8:1693-1696 (1993); and Brent, WO94/10300. Such variant protein-binding proteins are also likely to beinvolved in the propagation of signals by the variant proteins orvariant protein targets as, for example, elements of a protein-mediatedsignaling pathway. Alternatively, such variant protein-binding proteinsare inhibitors of the variant protein.

The two-hybrid system is based on the modular nature of mosttranscription factors, which typically consist of separable DNA-bindingand activation domains. Briefly, the assay typically utilizes twodifferent DNA constructs. In one construct, the gene that codes for avariant protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a variantprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) that is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detected,and cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene that encodes the proteinthat interacts with the variant protein.

Antibodies Directed to Variant Proteins

The present invention also provides antibodies that selectively bind tothe variant proteins disclosed herein and fragments thereof. Suchantibodies may be used to quantitatively or qualitatively detect thevariant proteins of the present invention. As used herein, an antibodyselectively binds a target variant protein when it binds the variantprotein and does not significantly bind to non-variant proteins, i.e.,the antibody does not significantly bind to normal, wild-type, orart-known proteins that do not contain a variant amino acid sequence dueto one or more SNPs of the present invention (variant amino acidsequences may be due to, for example, nonsynonymous cSNPs, nonsense SNPsthat create a stop codon, thereby causing a truncation of a polypeptideor SNPs that cause read-through mutations resulting in an extension of apolypeptide).

As used herein, an antibody is defined in terms consistent with thatrecognized in the art: they are multi-subunit proteins produced by anorganism in response to an antigen challenge. The antibodies of thepresent invention include both monoclonal antibodies and polyclonalantibodies, as well as antigen-reactive proteolytic fragments of suchantibodies, such as Fab, F(ab)′2, and Fv fragments. In addition, anantibody of the present invention further includes any of a variety ofengineered antigen-binding molecules such as a chimeric antibody (U.S.Pat. Nos. 4,816,567 and 4,816,397; Morrison et al., Proc Natl Acad SciUSA 81:6851 (1984); Neuberger et al., Nature 312:604 (1984)), ahumanized antibody (U.S. Pat. Nos. 5,693,762; 5,585,089 and 5,565,332),a single-chain Fv (U.S. Pat. No. 4,946,778; Ward et al., Nature 334:544(1989)), a bispecific antibody with two binding specificities (Segal etal., J Immunol Methods 248:1 (2001); Carter, J Immunol Methods 248:7(2001)), a diabody, a triabody, and a tetrabody (Todorovska et al., JImmunol Methods 248:47 (2001)), as well as a Fab conjugate (dimer ortrimer), and a minibody.

Many methods are known in the art for generating and/or identifyingantibodies to a given target antigen. Harlow, Antibodies, Cold SpringHarbor Press, N.Y. (1989). In general, an isolated peptide (e.g., avariant protein of the present invention) is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit, hamster ormouse. Either a full-length protein, an antigenic peptide fragment(e.g., a peptide fragment containing a region that varies between avariant protein and a corresponding wild-type protein), or a fusionprotein can be used. A protein used as an immunogen may benaturally-occurring, synthetic or recombinantly produced, and may beadministered in combination with an adjuvant, including but not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substance such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and the like.

Monoclonal antibodies can be produced by hybridoma technology, whichimmortalizes cells secreting a specific monoclonal antibody. Kohler andMilstein, Nature 256:495 (1975). The immortalized cell lines can becreated in vitro by fusing two different cell types, typicallylymphocytes, and tumor cells. The hybridoma cells may be cultivated invitro or in vivo. Additionally, fully human antibodies can be generatedby transgenic animals. He et al., J Immunol 169:595 (2002). Fd phage andFd phagemid technologies may be used to generate and select recombinantantibodies in vitro. Hoogenboom and Chames, Immunol Today 21:371 (2000);Liu et al., J Mol Biol 315:1063 (2002). The complementarity-determiningregions of an antibody can be identified, and synthetic peptidescorresponding to such regions may be used to mediate antigen binding.U.S. Pat. No. 5,637,677.

Antibodies are preferably prepared against regions or discrete fragmentsof a variant protein containing a variant amino acid sequence ascompared to the corresponding wild-type protein (e.g., a region of avariant protein that includes an amino acid encoded by a nonsynonymouscSNP, a region affected by truncation caused by a nonsense SNP thatcreates a stop codon, or a region resulting from the destruction of astop codon due to read-through mutation caused by a SNP). Furthermore,preferred regions will include those involved in function/activityand/or protein/binding partner interaction. Such fragments can beselected on a physical property, such as fragments corresponding toregions that are located on the surface of the protein, e.g.,hydrophilic regions, or can be selected based on sequence uniqueness, orbased on the position of the variant amino acid residue(s) encoded bythe SNPs provided by the present invention. An antigenic fragment willtypically comprise at least about 8-10 contiguous amino acid residues inwhich at least one of the amino acid residues is an amino acid affectedby a SNP disclosed herein. The antigenic peptide can comprise, however,at least 12, 14, 16, 20, 25, 50, 100 (or any other number in-between) ormore amino acid residues, provided that at least one amino acid isaffected by a SNP disclosed herein.

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody or an antigen-reactivefragment thereof to a detectable substance. Detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies, particularly the use of antibodies as therapeutic agents,are reviewed in: Morgan, “Antibody therapy for Alzheimer's disease,”Expert Rev Vaccines (1):53-9 (February 2003); Ross et al., “Anticancerantibodies,” Am J Clin Pathol 119(4):472-85 (April 2003); Goldenberg,“Advancing role of radiolabeled antibodies in the therapy of cancer,”Cancer Immunol Immunother 52(5):281-96 (May 2003); Epub Mar. 11, 2003;Ross et al., “Antibody-based therapeutics in oncology,” Expert RevAnticancer Ther 3(1):107-21 (February 2003); Cao et al., “Bispecificantibody conjugates in therapeutics,” Adv Drug Deliv Rev 55(2):171-97(February 2003); von Mehren et al., “Monoclonal antibody therapy forcancer,” Annu Rev Med 54:343-69 (2003); Epub Dec. 3, 2001; Hudson etal., “Engineered antibodies,” Nat Med 9(1):129-34 (January 2003); Brekkeet al., “Therapeutic antibodies for human diseases at the dawn of thetwenty-first century,” Nat Rev Drug Discov 2(1):52-62 (January 2003);Erratum in: Nat Rev Drug Discov 2(3):240 (March 2003); Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems,” Curr Opin Biotechnol 13(6):625-9 (December 2002); Andreakos etal., “Monoclonal antibodies in immune and inflammatory diseases,” CurrOpin Biotechnol 13(6):615-20 (December 2002); Kellermann et al.,“Antibody discovery: the use of transgenic mice to generate humanmonoclonal antibodies for therapeutics,” Curr Opin Biotechnol13(6):593-7 (December 2002); Pini et al., “Phage display and colonyfilter screening for high-throughput selection of antibody libraries,”Comb Chem High Throughput Screen 5(7):503-10 (November 2002); Batra etal., “Pharmacokinetics and biodistribution of genetically engineeredantibodies,” Curr Opin Biotechnol 13(6):603-8 (December 2002); andTangri et al., “Rationally engineered proteins or antibodies with absentor reduced immunogenicity,” Curr Med Chem 9(24):2191-9 (December 2002).

Uses of Antibodies

Antibodies can be used to isolate the variant proteins of the presentinvention from a natural cell source or from recombinant host cells bystandard techniques, such as affinity chromatography orimmunoprecipitation. In addition, antibodies are useful for detectingthe presence of a variant protein of the present invention in cells ortissues to determine the pattern of expression of the variant proteinamong various tissues in an organism and over the course of normaldevelopment or disease progression. Further, antibodies can be used todetect variant protein in situ, in vitro, in a bodily fluid, or in acell lysate or supernatant in order to evaluate the amount and patternof expression. Also, antibodies can be used to assess abnormal tissuedistribution, abnormal expression during development, or expression inan abnormal condition, such as in CVD, or during statin treatment.Additionally, antibody detection of circulating fragments of thefull-length variant protein can be used to identify turnover.

Antibodies to the variant proteins of the present invention are alsouseful in pharmacogenomic analysis. Thus, antibodies against variantproteins encoded by alternative SNP alleles can be used to identifyindividuals that require modified treatment modalities.

Further, antibodies can be used to assess expression of the variantprotein in disease states such as in active stages of the disease or inan individual with a predisposition to a disease related to theprotein's function, such as CVD, or during the course of a treatmentregime, such as during statin treatment. Antibodies specific for avariant protein encoded by a SNP-containing nucleic acid molecule of thepresent invention can be used to assay for the presence of the variantprotein, such as to determine an individual's response to statintreatment (particularly for reducing their risk for CVD, particularlyCHD, such as MI, or stroke) or to diagnose CVD orpredisposition/susceptibility to CVD, as indicated by the presence ofthe variant protein.

Antibodies are also useful as diagnostic tools for evaluating thevariant proteins in conjunction with analysis by electrophoreticmobility, isoelectric point, tryptic peptide digest, and other physicalassays well known in the art.

Antibodies are also useful for tissue typing. Thus, where a specificvariant protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

Antibodies can also be used to assess aberrant subcellular localizationof a variant protein in cells in various tissues. The diagnostic usescan be applied, not only in genetic testing, but also in monitoring atreatment modality. Accordingly, where treatment is ultimately aimed atcorrecting the expression level or the presence of variant protein oraberrant tissue distribution or developmental expression of a variantprotein, antibodies directed against the variant protein or relevantfragments can be used to monitor therapeutic efficacy.

The antibodies are also useful for inhibiting variant protein function,for example, by blocking the binding of a variant protein to a bindingpartner. These uses can also be applied in a therapeutic context inwhich treatment involves inhibiting a variant protein's function. Anantibody can be used, for example, to block or competitively inhibitbinding, thus modulating (agonizing or antagonizing) the activity of avariant protein. Antibodies can be prepared against specific variantprotein fragments containing sites required for function or against anintact variant protein that is associated with a cell or cell membrane.For in vivo administration, an antibody may be linked with an additionaltherapeutic payload such as a radionuclide, an enzyme, an immunogenicepitope, or a cytotoxic agent. Suitable cytotoxic agents include, butare not limited to, bacterial toxin such as diphtheria, and plant toxinsuch as ricin. The in vivo half-life of an antibody or a fragmentthereof may be lengthened by pegylation through conjugation topolyethylene glycol. Leong et al., Cytokine 16:106 (2001).

The invention also encompasses kits for using antibodies, such as kitsfor detecting the presence of a variant protein in a test sample. Anexemplary kit can comprise antibodies such as a labeled or labelableantibody and a compound or agent for detecting variant proteins in abiological sample; means for determining the amount, or presence/absenceof variant protein in the sample; means for comparing the amount ofvariant protein in the sample with a standard; and instructions for use.

Vectors and Host Cells

The present invention also provides vectors containing theSNP-containing nucleic acid molecules described herein. The term“vector” refers to a vehicle, preferably a nucleic acid molecule, whichcan transport a SNP-containing nucleic acid molecule. When the vector isa nucleic acid molecule, the SNP-containing nucleic acid molecule can becovalently linked to the vector nucleic acid. Such vectors include, butare not limited to, a plasmid, single or double stranded phage, a singleor double stranded RNA or DNA viral vector, or artificial chromosome,such as a BAC, PAC, YAC, or MAC.

A vector can be maintained in a host cell as an extrachromosomal elementwhere it replicates and produces additional copies of the SNP-containingnucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the SNP-containingnucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the SNP-containingnucleic acid molecules. The vectors can function in prokaryotic oreukaryotic cells or in both (shuttle vectors).

Expression vectors typically contain cis-acting regulatory regions thatare operably linked in the vector to the SNP-containing nucleic acidmolecules such that transcription of the SNP-containing nucleic acidmolecules is allowed in a host cell. The SNP-containing nucleic acidmolecules can also be introduced into the host cell with a separatenucleic acid molecule capable of affecting transcription. Thus, thesecond nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the SNP-containing nucleic acid molecules from thevector. Alternatively, a trans-acting factor may be supplied by the hostcell. Finally, a trans-acting factor can be produced from the vectoritself. It is understood, however, that in some embodiments,transcription and/or translation of the nucleic acid molecules can occurin a cell-free system.

The regulatory sequences to which the SNP-containing nucleic acidmolecules described herein can be operably linked include promoters fordirecting mRNA transcription. These include, but are not limited to, theleft promoter from bacteriophage λ, the lac, TRP, and TAC promoters fromE. coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region, aribosome-binding site for translation. Other regulatory control elementsfor expression include initiation and termination codons as well aspolyadenylation signals. A person of ordinary skill in the art would beaware of the numerous regulatory sequences that are useful in expressionvectors. See, e.g., Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (2000).

A variety of expression vectors can be used to express a SNP-containingnucleic acid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example, vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors can also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g., cosmids and phagemids. Appropriate cloning andexpression vectors for prokaryotic and eukaryotic hosts are described inSambrook and Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, N.Y. (2000).

The regulatory sequence in a vector may provide constitutive expressionin one or more host cells (e.g., tissue specific expression) or mayprovide for inducible expression in one or more cell types such as bytemperature, nutrient additive, or exogenous factor, e.g., a hormone orother ligand. A variety of vectors that provide constitutive orinducible expression of a nucleic acid sequence in prokaryotic andeukaryotic host cells are well known to those of ordinary skill in theart.

A SNP-containing nucleic acid molecule can be inserted into the vectorby methodology well-known in the art. Generally, the SNP-containingnucleic acid molecule that will ultimately be expressed is joined to anexpression vector by cleaving the SNP-containing nucleic acid moleculeand the expression vector with one or more restriction enzymes and thenligating the fragments together. Procedures for restriction enzymedigestion and ligation are well known to those of ordinary skill in theart.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial host cells include, but are notlimited to, Escherichia coli, Streptomyces spp., and Salmonellatyphimurium. Eukaryotic host cells include, but are not limited to,yeast, insect cells such as Drosophila spp., animal cells such as COSand CHO cells, and plant cells.

As described herein, it may be desirable to express the variant peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the variant peptides. Fusion vectorscan, for example, increase the expression of a recombinant protein,increase the solubility of the recombinant protein, and aid in thepurification of the protein by acting, for example, as a ligand foraffinity purification. A proteolytic cleavage site may be introduced atthe junction of the fusion moiety so that the desired variant peptidecan ultimately be separated from the fusion moiety. Proteolytic enzymessuitable for such use include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in a bacterial host byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein (S.Gottesman, Gene Expression Technology: Methods in Enzymology185:119-128, Academic Press, Calif. (1990)). Alternatively, the sequenceof the SNP-containing nucleic acid molecule of interest can be alteredto provide preferential codon usage for a specific host cell, forexample, E. coli. Wada et al., Nucleic Acids Res 20:2111-2118 (1992).

The SNP-containing nucleic acid molecules can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast (e.g., S. cerevisiae) include pYepSec1 (Baldari etal., EMBO J 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2(Invitrogen Corporation, San Diego, Calif.).

The SNP-containing nucleic acid molecules can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,Mol Cell Biol 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,Virology 170:31-39 (1989)).

In certain embodiments of the invention, the SNP-containing nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (B. Seed, Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J 6:187-195 (1987)).

The invention also encompasses vectors in which the SNP-containingnucleic acid molecules described herein are cloned into the vector inreverse orientation, but operably linked to a regulatory sequence thatpermits transcription of antisense RNA. Thus, an antisense transcriptcan be produced to the SNP-containing nucleic acid sequences describedherein, including both coding and non-coding regions. Expression of thisantisense RNA is subject to each of the parameters described above inrelation to expression of the sense RNA (regulatory sequences,constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include, for example,prokaryotic cells, lower eukaryotic cells such as yeast, othereukaryotic cells such as insect cells, and higher eukaryotic cells suchas mammalian cells.

The recombinant host cells can be prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to persons of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those described in Sambrook and Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, N.Y. (2000).

Host cells can contain more than one vector. Thus, differentSNP-containing nucleotide sequences can be introduced in differentvectors into the same cell. Similarly, the SNP-containing nucleic acidmolecules can be introduced either alone or with other nucleic acidmolecules that are not related to the SNP-containing nucleic acidmolecules, such as those providing trans-acting factors for expressionvectors. When more than one vector is introduced into a cell, thevectors can be introduced independently, co-introduced, or joined to thenucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication can occur in host cells that provide functionsthat complement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be inserted in the same vector that containsthe SNP-containing nucleic acid molecules described herein or may be ina separate vector. Markers include, for example, tetracycline orampicillin-resistance genes for prokaryotic host cells, anddihydrofolate reductase or neomycin resistance genes for eukaryotic hostcells. However, any marker that provides selection for a phenotypictrait can be effective.

While the mature variant proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these variant proteins using RNA derivedfrom the DNA constructs described herein.

Where secretion of the variant protein is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asG-protein-coupled receptors (GPCRs), appropriate secretion signals canbe incorporated into the vector. The signal sequence can be endogenousto the peptides or heterologous to these peptides.

Where the variant protein is not secreted into the medium, the proteincan be isolated from the host cell by standard disruption procedures,including freeze/thaw, sonication, mechanical disruption, use of lysingagents, and the like. The variant protein can then be recovered andpurified by well-known purification methods including, for example,ammonium sulfate precipitation, acid extraction, anion or cationicexchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that, depending upon the host cell in whichrecombinant production of the variant proteins described herein occurs,they can have various glycosylation patterns, or may benon-glycosylated, as when produced in bacteria. In addition, the variantproteins may include an initial modified methionine in some cases as aresult of a host-mediated process.

For further information regarding vectors and host cells, see CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y.

Uses of Vectors and Host Cells, and Transgenic Animals

Recombinant host cells that express the variant proteins describedherein have a variety of uses. For example, the cells are useful forproducing a variant protein that can be further purified into apreparation of desired amounts of the variant protein or fragmentsthereof. Thus, host cells containing expression vectors are useful forvariant protein production.

Host cells are also useful for conducting cell-based assays involvingthe variant protein or variant protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a variant protein is useful forassaying compounds that stimulate or inhibit variant protein function.Such an ability of a compound to modulate variant protein function maynot be apparent from assays of the compound on the native/wild-typeprotein, or from cell-free assays of the compound. Recombinant hostcells are also useful for assaying functional alterations in the variantproteins as compared with a known function.

Genetically-engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably anon-human mammal, for example, a rodent, such as a rat or mouse, inwhich one or more of the cells of the animal include a transgene. Atransgene is exogenous DNA containing a SNP of the present inventionwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal inone or more of its cell types or tissues. Such animals are useful forstudying the function of a variant protein in vivo, and identifying andevaluating modulators of variant protein activity. Other examples oftransgenic animals include, but are not limited to, non-human primates,sheep, dogs, cows, goats, chickens, and amphibians. Transgenic non-humanmammals such as cows and goats can be used to produce variant proteinswhich can be secreted in the animal's milk and then recovered.

A transgenic animal can be produced by introducing a SNP-containingnucleic acid molecule into the male pronuclei of a fertilized oocyte,e.g., by microinjection or retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any nucleic acidmolecules that contain one or more SNPs of the present invention canpotentially be introduced as a transgene into the genome of a non-humananimal.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the variant protein in particularcells or tissues.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al.; U.S. Pat. No.4,873,191 by Wagner et al., and in B. Hogan, Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, N.Y. (1986). Similarmethods are used for production of other transgenic animals. Atransgenic founder animal can be identified based upon the presence ofthe transgene in its genome and/or expression of transgenic mRNA intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene can further be bred to othertransgenic animals carrying other transgenes. A transgenic animal alsoincludes a non-human animal in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. Lakso et al., PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae. O'Gorman et al., Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are generally needed. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected variant protein and the other containing a transgeneencoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described, for example, in I. Wilmutet al., Nature 385:810-813 (1997) and PCT International Publication Nos.WO 97/07668 and WO 97/07669. In brief, a cell (e.g., a somatic cell)from the transgenic animal can be isolated and induced to exit thegrowth cycle and enter G_(o) phase. The quiescent cell can then befused, e.g., through the use of electrical pulses, to an enucleatedoocyte from an animal of the same species from which the quiescent cellis isolated. The reconstructed oocyte is then cultured such that itdevelops to morula or blastocyst and then transferred to pseudopregnantfemale foster animal. The offspring born of this female foster animalwill be a clone of the animal from which the cell (e.g., a somatic cell)is isolated.

Transgenic animals containing recombinant cells that express the variantproteins described herein are useful for conducting the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could influence ligand orsubstrate binding, variant protein activation, signal transduction, orother processes or interactions, may not be evident from in vitrocell-free or cell-based assays. Thus, non-human transgenic animals ofthe present invention may be used to assay in vivo variant proteinfunction as well as the activities of a therapeutic agent or compoundthat modulates variant protein function/activity or expression. Suchanimals are also suitable for assessing the effects of null mutations(i.e., mutations that substantially or completely eliminate one or morevariant protein functions).

For further information regarding transgenic animals, see Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems,” Curr Opin Biotechnol 13(6):625-9 (December 2002); Petters etal., “Transgenic animals as models for human disease,” Transgenic Res9(4-5):347-51, discussion 345-6 (2000); Wolf et al., “Use of transgenicanimals in understanding molecular mechanisms of toxicity,” J PharmPharmacol 50(6):567-74 (June 1998); Echelard, “Recombinant proteinproduction in transgenic animals,” Curr Opin Biotechnol 7(5):536-40(October 1996); Houdebine, “Transgenic animal bioreactors,” TransgenicRes 9(4-5):305-20 (2000); Pirity et al., “Embryonic stem cells, creatingtransgenic animals,” Methods Cell Biol 57:279-93 (1998); and Robl etal., “Artificial chromosome vectors and expression of complex proteinsin transgenic animals,” Theriogenology 59(1):107-13 (January 2003).

EXAMPLES

The following examples are offered to illustrate, but not limit, theclaimed invention.

Example 1: SNPs Associated with Statin Response in CARE, WOSCOPS, andPROVE IT-TIMI 22

Overview

In the study described here in Example 1, cohort and case-only studydesigns were used to identify SNPs associated with response to statintreatment. The entire cohort (individuals with and without incident CHDor CVD events) or cases only (only individuals with an incident CHD orCVD event) were analyzed in sample sets from the CARE, WOSCOPS, andPROVE IT. Specifically, analyses were carried out using these threesample sets to identify SNPs associated with a reduction in the risk ofCHD or CVD (CVD includes CHD and stroke), the results of which areprovided in Tables 4-7 and Tables 9-18 (Tables 9-18 provide additionalgenotyped SNPs as well as imputed SNPs).

Tables 4-7 provide results of analyses of statin response for either CHDor CVD reduction, in three genetic models (dominant, recessive, andadditive). Tables 4-7 provide SNPs that had a synergy index (odds ratio)with P value lower than 10⁻⁴ in a meta-analysis of CARE and WOSCOPScombined (Table 4-5) or in a meta-analysis of CARE, WOSCOPS, andPROVE-IT combined (Table 6-7), in any genetic model in either the CHD orCVD endpoint. Tables 4-5 provide meta-analyses of CARE and WOSCOPScombined, as well as logistic regression analysis of each sample setindividually. Tables 6-7 provide meta-analyses of CARE, WOSCOPS, andPROVE-IT combined, as well as logistic regression analysis of eachsample set individually.

Tables 5 and 7 provide analyses of certain LD SNPs in CARE and WOSCOPS(Table 5) and in CARE, WOSCOPS, and PROVE-IT (Table 7). For some SNPs,case-only data was available for a first SNP while cohort data wasavailable for a SNP in LD with the first SNP (LD SNP), which occurredwhen a working kPCR assay could not be made for the first SNP. For theseSNPs, the data for case-only analysis and the available data for thecohort is reported. The meta-analysis was performed with the cohort datawhen available. These SNPs are listed in Tables 5 and 7, with the twoSNPs in LD listed one below the other, and the degree of LD (r²) betweeneach of these pairs of SNPs is provided in Table 8.

CARE, WOSCOPS, and PROVE IT-TIMI 22 Sample Sets

The CARE (“Cholesterol and Recurrent Events”) and WOSCOPS (“West ofScotland Coronary Prevention Study”) studies were prospective trialsthat assessed the effect of pravastatin (40 mg/day) on the prevention ofMI and CHD. CARE was a secondary prevention trial and WOSCOPS was aprimary prevention trial. The PROVE IT-TIMI 22 (“Pravastatin orAtorvastatin Evaluation and Infection Therapy: Thrombolysis inMyocardial Infarction 22”; which is interchangeably referred to hereinas “PROVE-IT”) trial evaluated the effectiveness of intensive therapywith high-dose atorvastatin (80 mg/day) versus moderate therapy withstandard-dose pravastatin (40 mg/day, which was the dose used in theCARE and WOSCOPS trials) in preventing death or cardiovascular events inpatients with a recent acute coronary syndrome.

These trials and the sample sets from these trials (such as theinclusion criteria for participants) are described in the followingreferences. Those portions of each of the following references thatpertain to the CARE, WOSCOPS, and PROVE-IT trials and sample sets arehereby incorporated by reference. CARE is described in Sacks et al.,“Cholesterol and Recurrent Events Trial Investigators. The effect ofpravastatin on coronary events after myocardial infarction in patientswith average cholesterol levels”, N Engl J Med 1996; 335:1001-9, andWOSCOPS is described in Shepherd et al., “West of Scotland CoronaryPrevention Study Group. Prevention of coronary heart disease withpravastatin in men with hypercholesterolemia”, N Engl J Med 1995;333:1301-7. PROVE-IT is described in Iakoubova et al., Polymorphism inKIF6 gene and benefit from statins after acute coronary syndromes:results from the PROVE IT-TIMI 22 study”, J Am Coll Cardiol. 2008 Jan.29; 51(4):449-55 and Cannon et al., “Intensive versus moderate lipidlowering with statins after acute coronary syndromes”, N Engl J Med2004; 350:1495-504.

Endpoints

The endpoint definitions used in these analyses of CARE, WOSCOPS, andPROVE-IT (the results of which are provided in Tables 4-7) were asfollows. The CHD endpoint was defined in the analyses herein of CARE asa composite endpoint of fatal CHD, definite non-fatal MI, orrevascularization, and was defined in the analyses herein of WOSCOPS asa composite endpoint of death from CHD, nonfatal MI, orrevascularization. In both the CARE and WOSCOPS analyses herein, the CVDendpoint was defined as a composite endpoint of CHD or stroke. Theanalyses herein of PROVE-IT analyzed the primary endpoint of PROVE-IT,which was a composite endpoint of revascularization (if performed atleast 30 days after randomization), unstable angina requiringhospitalization, MI, all causes of death, or stroke. Thus, there wasonly one endpoint for PROVE-IT (the composite primary endpoint of theoriginal PROVE-IT study, which includes some stroke cases), and thisendpoint was used in the meta-analysis for both CHD and CVD provided inTables 6-7. With respect to stroke, in the analyses herein of CARE andPROVE-IT, stroke was defined as stroke or transient ischemic attack(TIA), and in the analyses herein of WOSCOPS, stroke was defined asfatal or non-fatal stroke. Revascularization, which can includepercutaneous transluminal coronary angioplasty (PTCA), stent placement,and coronary artery bypass graft (CABG), are medical interventions thatindicate the presence of CHD.

Study Designs

Cohort and case-only study designs were used to identify SNPs associatedwith response to statin treatment. The entire cohort (individuals withand without incident CHD or CVD events; identified as “cohort” in the“Source” column of Tables 4-7) or only individuals with an incident CHDor CVD event (identified as “CaseOnly” in the “Source” column of Tables4-7) were analyzed in sample sets from the CARE, WOSCOPS, and PROVE-ITtrials to test whether the reduction of CHD/CVD events by statin therapy(for CARE and WOSCOPS studies), or by high dose atorvastatin therapy(for the PROVE IT study), differed according to genotype (a treatment bySNP interaction) for each SNP evaluated in the study.

For each SNP, a logistic regression model having treatment status as thedependent variable and SNP as the independent predictor variable wasperformed, with terms for age, sex and race included in the model ascovariates. The anti-log of the regression coefficient corresponding tothe SNP is an estimate of the synergy index (SI) (Davis et al.,“Imputing gene-treatment interactions when the genotype distribution isunknown using case-only and putative placebo analyses—a new method forthe Genetics of Hypertension Associated Treatment (GenHAT) study”,Statistics in Medicine 23: (2004), pages 2413-2427). The SI is a ratioof odds ratios: for example in the CARE and WOSCOPS studies, the SIrepresents the factor by which the odds-ratio of statin treatment,compared with placebo, among major homozygous individuals is multipliedby in order to obtain the odds-ratio of treatment vs. placebo amongheterozygous individuals; and multiplied by a second time to obtain theodds-ratio of treatment vs. placebo in minor homozygous individuals. Thecase-only study design results in a valid estimate of the SI under theassumption that genotype and treatment are independent in thepopulation. In a randomized clinical trial, genotype and treatment areindependent by design. The p-value for the regression coefficientcorresponding to the SNP results from a test of the null hypothesis thatthe regression coefficient is equal to zero (SI is equal to one) andthus small p-values indicate the SI is unlikely equal to one and thatthe effect of treatment likely differs by genotype.

The logistic regression models were performed separately for each ofCARE, WOSCOPS, and PROVE-IT in order to obtain study-specific results. Ameta-analysis was then used to estimate the combined evidence forinteraction when considering either the CARE and WOSCOPS studies (Tables4-5), or all three studies (CARE, WOSCOPS, and PROVE-IT) (Tables 6-7).The meta-analysis used the inverse variance method (Rothman et al.,1998; Modern Epidemiology, 2nd edition, Lippincott Williams & Wilkins,Philadelphia, PA, pages 660-661) to calculate the combined SI using aweighted average of the effects of the individual studies with weightsequal to the inverse variance from each study.

The logistic regression and meta-analyses were performed using PLINKversion 1.07 (Purcell et al. (2007), “PLINK: A tool set for whole-genomeassociation and population-based linkage analyses”, Am. J. Hum. Genet.81, 559-575).

Regarding case-only study designs specifically, further informationabout these study designs is provided in Piegorsch et al.,“Non-hierarchical logistic models and case-only designs for assessingsusceptibility in population-based case-control studies”, Statistics inMedicine 13 (1994) (pages 153-162); Khoury et al., “NontraditionalEpidemiologic Approaches in the Analysis of Gene-EnvironmentInteraction: Case-Control Studies with No Controls!”, American Journalof Epidemiology 144:3 (1996) (pages 207-213); Pierce et al., “Case-onlygenome-wide interaction study of disease risk, prognosis and treatment”,Genet Epidemiol. 2010 January; 34(1):7-15; Begg et al., “Statisticalanalysis of molecular epidemiology studies employing case-series”,Cancer Epidemiology Biomarkers and Prevention 3 (1994) pp 173-1′75; Yanget al., “Sample Size Requirements in Case-Only Designs to DetectGene-Environment Interaction”, American Journal of Epidemiology 146:9(1997) pp 713-720; Albert et al., “Limitations of the Case-only Designfor Identifying Gene-Environment Interactions”, American Journal ofEpidemiology 154:8 (2001) pp 687-693; and Wang et al., “PopulationStratification Bias in the Case-Only Study for Gene-EnvironmentInteractions”, American Journal of Epidemiology 168:2 (2008) pp 197-201,each of which is incorporated herein by reference in its entirety.Further information about genome-wide association studies is provided inWellcome Trust Case Control Consortium, “Genome-wide association studyof 14,000 cases of seven common diseases and 3,000 shared controls”,Nature. 2007 Jun. 7; 447(7145):661-78 and Ikram et al., “Genomewideassociation studies of stroke”, N Engl J Med. 2009 Apr. 23;360(17):1718-28.

Identification of Additional Statin Response-Associated SNPs byImputation and Genotyping

Additional genotyped and imputed SNPs were identified as beingassociated with statin response in the CARE, WOSCOPS, and PROVE-ITsample sets, and these additional SNPs are provided in Tables 9-18. Theassociation of certain of these SNPs with statin response was identifiedby genotyping, whereas the association of certain other SNPs with statinresponse was identified by imputation. Imputation involves imputing theallele/genotype present at a SNP for each individual in the sample set(CARE, WOSCOPS, and PROVE-IT) rather than directly genotyping the SNP ina sample from the individual. Thus, Tables 9-18 include SNPs identifiedby imputation as well as SNPs identified by genotyping, and the columnlabeled “Source” in Tables 9-18 indicates whether each SNP was genotypedor imputed (all of the SNPs provided in Tables 4-7 were identified bygenotyping).

Specifically, Tables 9-18 provide SNPs for which the p-value for arandom effect was lower than 10⁻⁴ for either the meta-analysis of CAREand WOSCOPS combined or the meta-analysis of CARE, WOSCOPS, and PROVE-ITcombined, for either the CHD or CVD endpoint, and for any genetic model(dominant, recessive, additive, or genotypic). Association interactionbetween statin response and either the CHD or CVD phenotype wasperformed. SNPs were either imputed or genotyped.

Imputation was carried out using the BEAGLE genetic analysis program toanalyze genotyping data from the HapMap project (The InternationalHapMap Consortium, NCBI, NLM, NIH). Imputation and the BEAGLE program(including the modeling algorithm that BEAGLE utilizes) are described inthe following references: Browning, “Missing data imputation andhaplotype phase inference for genome-wide association studies”, HumGenet (2008) 124:439-450 (which reviews imputation and BEAGLE); B LBrowning and S R Browning (2009) “A unified approach to genotypeimputation and haplotype phase inference for large data sets of triosand unrelated individuals”. Am J Hum Genet 84:210-223 (which describesBEAGLE's methods for imputing ungenotyped markers and phasingparent-offspring trios); S R Browning and B L Browning (2007) “Rapid andaccurate haplotype phasing and missing data inference for whole genomeassociation studies using localized haplotype clustering”. Am J HumGenet 81:1084-1097 (which describes BEAGLE's methods for inferringhaplotype phase or sporadic missing data in unrelated individuals); B LBrowning and S R Browning (2007) “Efficient multilocus associationmapping for whole genome association studies using localized haplotypeclustering”. Genet Epidemiol 31:365-375 (which describes BEAGLE'smethods for association testing); S R Browning (2006) “Multilocusassociation mapping using variable-length Markov chains”. Am J Hum Genet78:903-13 (which describes BEAGLE's haplotype frequency model); and B LBrowning and S R Browning (2008) “Haplotypic analysis of Wellcome TrustCase Control Consortium data”. Human Genetics 123:273-280 (whichdescribes an example in which BEAGLE was used to analyze a largegenome-wide association study). Each of these references related toimputation and the BEAGLE program is incorporated herein by reference inits entirety.

Example 2: Polymorphism Rs11556924 in the ZC3HC1 Gene is Associated withDifferential CHD Risk Reduction by Statin Therapy in CARE and WOSCOPS

A case-only study design was used to test whether the reduction of CHDevents by statin therapy (for CARE and WOSCOPS studies) differedaccording to genotype (a treatment by SNP interaction) for each SNPevaluated in the study.

Herein in Example 2, SNPs previously reported to be associated withcoronary artery disease (Schunkert et al., “Large-scale associationanalysis identifies 13 new susceptibility loci for coronary arterydisease”, Nat Genet. 2011 Mar. 6; and Peden et al., “A genome-wideassociation study in Europeans and South Asians identifies five new locifor coronary artery disease”, Nat Genet. 2011 Mar. 6) were analyzedusing the same methodology as described above in Example 1 in order todetermine whether any of these SNP are associated with differential CHDrisk reduction by statin therapy in a genome wide association studyconducted among cases of CARE and WOSCOPS.

It was determined from this analysis that SNP rs11556924 (hCV31283062)in the ZC3HC1 gene is associated with differential reduction of CHD riskby pravastatin therapy in both CARE and WOSCOPS (see Table 19).

Example 3: SNPs Around Chromosomal Locations 9p21 and 12p13 (NINJ2 andB4GALNT3 Gene Region) Associated with Stroke Statin Response and/orStroke Risk

Example 3 relates to genetic polymorphisms that are associated withstroke risk and/or stroke statin response (reduction of stroke risk bystatin treatment) (Tables 20-21) and CHD statin response (Table 22).

Table 20 provides SNPs associated with stroke risk and/or stroke statinresponse in the CARE sample set. For example, SNPs rs10757278 andrs1333049 at chromosomal location 9p21 were associated with a reductionof stroke events by statin treatment in CARE, particularly forheterozygotes (see Table 20). Furthermore, SNPs rs12425791 andrs11833579 at chromosomal location 12p13 near the NINJ2 gene wereassociated with stroke risk in the placebo arm of CARE (see Table 20).SNPs rs12425791 and rs11833579 were also associated with stroke statinresponse in that the homozygous and heterozygous carriers of either ofthese SNPs (i.e., carriers of the ‘A’ allele for either rs12425791 orrs11833579) had a greater reduction in stroke events with statintreatment compared with noncarriers (see Table 20). Consistent with theCARE trial, the stroke endpoint in the analysis for which the resultsare provided in Tables 20-21 included stroke as well as transientischemic attack (TIA).

Fine-mapping at the chromosome 12p13 locus was carried out by selecting77 tagging SNPs from a 400 kb region of the chromosome 12p13 locus whichcovered the NINJ2 gene and other genes, genotyping these 77 SNPs, andfurther imputing the genotypes of approximately 250 additional SNPs inthis region, for individuals in the CARE study. Analyzing thesefine-mapping SNPs for association with stroke risk in the placebo arm ofCARE and for stroke statin response in CARE identified SNP rs873134 inthe B4GALNT3 gene in the chromosome 12p13 region near NINJ2 (see Table21).

Table 22 provides results of an analysis of CHD statin response in CARE.Table 22 shows that SNP rs873134 is associated with response to statintreatment for reducing the risk of CHD (as well as for reducing the riskof stroke, as shown in Table 21). Specifically, Table 22 shows that SNPrs873134 is associated with a reduced occurrence of recurrent MI inindividuals in the CARE study who were treated with statins. Thus, SNPrs873134 is an example of a SNP that is associated with statin responsefor reducing risk for both stroke and CHD. In the analysis for which theresults are provided in Table 22, the endpoint was recurrent MI, and theanalysis was adjusted for age, gender, hypertension, diabetes, base LDLand HDL, and whether an individual was a current smoker.

Example 4: LD SNPs Associated with Statin Response and CVD

Another investigation was conducted to identify additional SNPs that arein high linkage disequilibrium (LD) with certain “interrogated SNPs”that have been found to be associated with response to statin treatment(particularly for reducing the risk of CVD, especially CHD such as MI).The “interrogated SNPs” were those SNPs provided in Tables 4-22 (theinterrogated SNPs are shown in columns 1-2 of Table 3, which indicatesthe hCV and rs identification numbers of each interrogated SNP), and theLD SNPs which were identified as being in high LD are provided in Table3 (in the columns labeled “LD SNP”, which indicate the hCV and rsidentification numbers of each LD SNP).

Specifically, Table 3 provides LD SNPs from the HapMap database (NCBI,NLM, NIH) that have linkage disequilibrium r² values of at least 0.9(the threshold r² value, which may also be designated as r_(T) ²) withan interrogated SNP. Each of these LD SNPs from the HapMap database iswithin 500 kb of its respective interrogated SNP, and the r² values arecalculated based on genotypes of HapMap Caucasian subjects. If aninterrogated SNP is not in the HapMap database, then there will not beany LD SNPs listed in Table 3 for that interrogated SNP.

As an example in Table 3, the interrogated SNP rs688358 (hCV1056543) wascalculated to be in LD with rs675163 (hCV1056544) at an r² value of 1(which is above the threshold r² value of 0.9), thus establishing thelatter SNP as a marker associated with statin response as well.

In this example, the threshold r² value was set at 0.9. However, thethreshold r² value can be set at other values such that one of ordinaryskill in the art would consider that any two SNPs having an r² valuegreater than or equal to the threshold r² value would be in sufficientLD with each other such that either SNP is useful for the sameutilities, such as determining an individual's response to statintreatment. For example, in various embodiments, the threshold r² valueused to classify SNPs as being in sufficient LD with an interrogated SNP(such that these LD SNPs can be used for the same utilities as theinterrogated SNP, for example) can be set at, for example, 0.7, 0.75,0.8, 0.85, 0.95, 0.96, 0.97, 0.98, 0.99, 1, etc. (or any other thresholdr² value in-between these values). Threshold r² values may be utilizedwith or without considering power or other calculations.

Sequences, SNP information, and associated gene/transcript/proteininformation for each of the LD SNPs listed in Table 3 is provided inTables 1-2. Thus, for any LD SNP listed in Table 3, sequence and alleleinformation (or other information) can be found by searching Tables 1-2using the hCV or rs identification number of the LD SNP of interest.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Modifications andvariations of the described compositions, methods and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the above-described modes for carryingout the invention that are obvious to those skilled in the field ofmolecular biology, genetics and related fields are intended to be withinthe scope of the following claims.

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LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US11827937B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method for reducing risk of coronary heartdisease (CHD) in a human, the method comprising: a) receiving anidentification of a human as being responsive to pravastatin treatmentfor reducing their CHD risk due to having a polymorphism rs3732788comprising C at position 101 of SEQ ID NO:903 or G at its complement;and b) administering pravastatin to said human.
 2. The method of claim1, wherein said human is homozygous for said C or said G.
 3. The methodof claim 1, wherein said human is heterozygous for said C or said G. 4.The method of claim 1, wherein said CHD is myocardial infarction.